Alternative workflows for plant proteomic analysis

High-throughput separations are intrinsic to the detection and analysis of peptides and proteins by mass spectrometry (MS). Together, efficient separation and MS can lead to the identification of thousands of proteins in a sample, cell or tissue and help build proteome maps that can be used to define a cell type or cellular state. Although 2D gels have been successfully used to separate proteins for subsequent MS analysis, alternative separation efficiencies and, consequently deeper results could be obtained with HPLC or other separation techniques that improve throughput. This highlight is aimed toward plant scientists who have special separation needs due to the nature of plant cells and who could benefit from knowing options and requirements for adopting alternative separation protocols. Through the various sample processing and protein separation strategies, plant biologists should be able to improve the quality of their proteomic reference maps and gain new information about the proteins that define plant cells.     

New genes and new biological roles for expansins

Expansins are extracellular proteins that loosen plant cell walls in novel ways. They are thought to function in cell enlargement, pollen tube invasion of the stigma (in grasses), wall disassembly during fruit ripening, abscission and other cell separation events. Expansins are encoded by two multigene families and each gene is often expressed in highly specific locations and cell types. Structural analysis indicates that one expansin region resembles the catalytic domain of family-45 endoglucanases but glucanase activity has not been detected. The genome projects have revealed numerous expansin-related sequences but their putative wall-loosening functions remain to be assessed.     

The proteomics of plant cell membranes

Membrane proteins are involved in many different functions depending on their location in the cell. Characterization of the membrane proteome can bring new insights to the function of different plant membrane systems and the subcellular compartments where the proteins are found. Plant membrane proteomics can also provide valuable information about plant-specific biological processes. Despite recent advances in the separation and techniques for the analysis of plant membrane proteins, characterization of these proteins, especially the hydrophobic ones, is still challenging. In this review, plant membrane proteomics data, compiled from the literature on Arabidopsis thaliana, are described. In addition, initial attempts towards determining the physiological significance of some proteins identified from membrane proteomics in rice are also described.     

What makes plants different? Principles of extracellular matrix function in 'soft' plant tissues

An overview of the biomechanic and morphogenetic function of the plant extracellular matrix (ECM) in its primary state is given. ECMs can play a pivotal role in cellular osmo- and volume-regulation, if they enclose the cell hermetically and constrain hydrostatic pressure evoked by osmotic gradients between the cell and its environment. From an engineering viewpoint, such cell walls turn cells into hydraulic machines, which establishes a crucial functional differences between cell walls and other cellular surface structures. Examples of such hydraulic machineries are discussed. The function of cell walls in the control of pressure, volume, and shape establishes constructional evolutionary constraints, which can explain aspects commonly considered typical of plants (sessility, autotrophy). In plants, 'cell division' by insertion of a new cell wall is a process of internal cytoplasmic differentiation. As such it differs fundamentally from cell separation during cytokinesis in animals, by leaving the coherence of the dividing protoplast basically intact. The resulting symplastic coherence appears more important for plant morphogenesis than histological structure; similar morphologies are realized on the basis of distinct tissue architectures in different plant taxa. The shape of a plant cell is determined by the shape its cell wall attains under multiaxial tensile stress. Consequently, the development of form in plants is achieved by a differential plastic deformation of the complex ECM in response to this multiaxial force (hydrostatic pressure). Current concepts of the regulation of these deformation processes are briefly evaluated.     

Novel nuclear protein ALC-INTERACTING PROTEIN1 is expressed in vascular and mesocarp cells in Arabidopsis

Pod shattering is an agronomical trait that is a result of the coordinated action of cell differentiation and separation. In Arabidopsis, pod shattering is controlled by a complex genetic network in which ALCATRAZ (ALC), a member of the basic helix-loop-helix family, is critical for cell separation during fruit dehiscence. Herein, we report the identification of ALC-INTERACTiNG PROTEIN1 (ACI1) via the yeast two-hybrid screen. ACI1 encodes a nuclear protein with a lysine-rich domain and a C-terminal serine-rich domain. ACI1 is mainly expressed in the vascular system throughout the plant and mesocarp of the valve in siliques. Our data showed that ACI1 interacts strongly with the N-terminal portion of ALC in yeast cells and in plant cells in the nucleus as demonstrated by bimolecular fluorescence complementation assay. Both ACl1 and ALC share an overlapping expression pattern, suggesting that they likely function together in planta. However, no detectable phenotype was found in plants with reduced ACI1 expression by RNA interference technology, suggesting that ACI1 may be redundant. Taken together, these data indicate that ALC may interact with ACll and its homologs to control cell separation during fruit dehiscence in Arabidopsis.     

Intercellular separation forces generated by intracellular pressure

Turgor pressure tends to force plant cells towards a spherical form, thus separating them at the angles from adjacent cells. In cooked vegetables containing starch, the swelling pressure of starch gelatinization generates analogous cell separation forces. A theoretical analysis of the relationship between internal pressure and cell separation forces is presented. Apart from the effect of internal pressure, cell separation forces increase with the diameter of the cell and decrease with the number of cell sides. Cell separation forces are reduced by the introduction of intercellular spaces and decrease further as these expand. The relationship between intracellular pressure and cell separation forces provides a basis upon which the strength of intercellular adhesion can be measured by experiment.     

Chromatography on cells and biomolecular assemblies

Red cells, biomembrane vesicles, proteoliposomes and liposomes non-covalently immobilized in gel particles or beads have been used as stationary phases for biomembrane affinity analyses and ion-exchange chromatographic separation. Lipid monolayers coupled to silica beads have been utilized for membrane protein purification in detergent solution and plant cell walls for group separation of macromolecules according to size and charge. Further methodological studies are essential to implement general practical application.     

Designer Condensates: A Toolkit for the Biomolecular Architect

Protein phase separation has emerged as a novel paradigm to explain the biogenesis of membraneless organelles and other so-called biomolecular condensates. While the implication of this physical phenomenon within cell biology is providing us with novel ways for understanding how cells compartmentalize biochemical reactions and encode function in such liquid-like assemblies, the newfound appreciation of this process also provides immense opportunities for designing and sculpting biological matter. Here, we propose that understanding the cell’s instruction manual of phase separation will enable bioengineers to begin creating novel functionalized biological materials and unprecedented tools for synthetic biology. We present FASE as the synthesis of the existing sticker-spacer framework, which explains the physical driving forces underlying phase separation, with quintessential principles of Scandinavian design. FASE serves both as a designer condensates catalogue and construction manual for the aspiring (membraneless) biomolecular architect. Our approach aims to inspire a new generation of bioengineers to rethink phase separation as an opportunity for creating reactive biomaterials with unconventional properties and to encode novel biological function in living systems. Although still in its infancy, several studies highlight how designer condensates have immediate and widespread potential applications in industry and medicine.     

Occludin localizes to centrosomes and modifies mitotic entry

Proper control of cell cycle progression and barrier function are essential processes to the maintenance of epithelial cell homeostasis. The contribution of tight junction proteins to barrier function is well established, whereas their contribution to cell cycle control is only beginning to be understood. Centrosomes are the principal microtubule organizing centers in eukaryotic cells and centrosome duplication and separation are linked to the cell cycle and mitotic entry. Here we demonstrate that occludin localizes with centrosomes in Madin-Darby canine kidney cells. Immunocytochemistry and biochemical fractionation studies reveal occludin localizes with centrosomes during interphase and occludin Ser-490 phosphorylation at centrosomes increases with mitotic entry. Stable expression of aspartic acid phosphomimetic (S490D) results in centrosomal localization of occludin and increases cell numbers. Furthermore, we provide evidence that occludin regulates centrosome separation and mitotic entry as the nonphosphorylatable alanine mutation (S490A) impedes centrosome separation, delays mitotic entry, and reduces proliferation. Collectively, these studies demonstrate a novel location and function for occludin in centrosome separation and mitosis.     

Separation and purification of plant terpenoids from biotransformation

The production of plant terpenoids through biotransformation has undoubtedly become one of the research hotspots, and the continuous upgrading of the corresponding downstream technology is also particularly important. Downstream technology is the indispensable technical channel for the industrialization of plant terpenoids. How to efficiently separate high‐purity products from complex microbial fermentation broths or enzyme‐catalyzed reactions to achieve high separation rates, high returns and environmental friendliness has become the focus of research in recent years. This review mainly introduces the common separation methods of plant terpenoids based on biotransformation from the perspectives of engineering strain construction, unit separation technology, product properties and added value. Then, further attention was paid to the application prospects of intelligent cell factories and control in the separation of plant terpenoids. Finally, some current challenges and prospects are proposed, which provide possible directions and guidance for the separation and purification of terpenoids and even industrialization.     

The Budding Yeast Amphiphysin Complex Is Required for Contractile Actin Ring (CAR) Assembly and Post-Contraction GEF-Independent Accumulation of Rho1-GTP

The late events of the budding yeast cell division cycle, cytokinesis and cell separation, require the assembly of a contractile actomyosin ring (CAR), primary and secondary septum formation followed by enzymatic degradation of the primary septum. Here we present evidence that demonstrates a role for the budding yeast amphiphysin complex, a heterodimer comprising Rvs167 and Rvs161, in CAR assembly and cell separation. The iqg1-1 allele is synthetically lethal with both rvs167 and rvs161 null mutations. We show that both Iqg1 and the amphiphysin complex are required for CAR assembly in early anaphase but cells are able to complete assembly in late anaphase when these activities are, respectively, either compromised or absent. Amphiphysin dependent CAR assembly is dependent upon the Rvs167 SH3 domain, but this function is insufficient to explain the observed synthetic lethality. Dosage suppression of the iqg1-1 allele demonstrates that endocytosis is required for the default cell separation pathway in the absence of CAR contraction but is unlikely to be required to maintain viability. The amphiphysin complex is required for normal, post-mitotic, localization of Chs3 and the Rho1 GEF, Rom2, which are responsible for secondary septum deposition and the accumulation of GTP bound Rho1 at the bud neck. It is concluded that a failure of polarity establishment in the absence of CAR contraction and amphiphysin function leads to loss of viability as a result of the consequent cell separation defect.     

Polygalacturonase45 cleaves pectin and links cell proliferation and morphogenesis to leaf curvature in Arabidopsis thaliana

Regulating plant architecture is a major goal in current breeding programs. Previous studies have increased our understanding of the genetic regulation of plant architecture, but it is also essential to understand how organ morphology is controlled at the cellular level. In the cell wall, pectin modification and degradation are required for organ morphogenesis, and these processes involve a series of pectin-modifying enzymes. Polygalacturonases (PGs) are a major group of pectin-hydrolyzing enzymes that cleave pectin backbones and release oligogalacturonides (OGs). PG genes function in cell expansion and separation, and contribute to organ expansion, separation and dehiscence in plants. However, whether and how they influence other cellular processes and organ morphogenesis are poorly understood. Here, we characterized the functions of Arabidopsis PG45 (PG45) in organ morphogenesis using genetic, developmental, cell biological and biochemical analyses. A heterologously expressed portion of PG45 cleaves pectic homogalacturonan in vitro, indicating that PG45 is a bona fide PG. PG45 functions in leaf and flower structure, branch formation and organ growth. Undulation in pg45 knockout and PG45 overexpression leaves is accompanied by impaired adaxial–abaxial polarity, and loss of PG45 shortens the duration of cell proliferation in the adaxial epidermis of developing leaves. Abnormal leaf curvature is coupled with altered pectin metabolism and autogenous OG profiles in pg45 knockout and PG45 overexpression leaves. Together, these results highlight a previously underappreciated function for PGs in determining tissue polarity and regulating cell proliferation, and imply the existence of OG-based signaling pathways that modulate plant development.     

Identification of a putative receptor-ligand pair controlling cell separation in plants

Cell separation events are important throughout the lifespan of a plant. To assure that the plant''s integrity is not compromised, such events, which depend on cell wall degradation, have to be tightly controlled both in time and space. The final step of floral organ abscission in Arabidopsis is controlled by INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), in that mutation of IDA causes a block in abscission. Overexpression results in early abscission of floral organs. In a recent article we show that this is also the case when overexpressing the related IDA-LIKE (IDL) proteins, indicating a degree of functional redundancy. Based on gene swap and deletion constructs introduced in the ida mutant and synthetic peptide assays we demonstrated that the conserved C-terminal motif (EPIP) of IDA and IDL1 was sufficient to replace IDA function. This function is dependent on the presence of the receptor-like kinases (RLK) HAESA (HAE) and HAESA-LIKE2 (HSL2), suggesting that an IDA peptide acts as a ligand interacting with these receptors. Our study further revealed that the five IDL genes are expressed at various sites where cell separation takes place. We suggest that the IDL proteins constitute a family of ligands that act through RLKs similar to HAESA and control cell separation at different sites and development stages during the life of the plant.Key words: Arabidopsis, signaling, receptor, ligand, gene-family, cell separation, HAE, IDA     

Separation of CHO cells using hydrocyclones

Hydrocyclones are simple and robust separation devices with no moving parts. In the past few years, their use in animal cell separation has been proposed. In this work, the use of different hydrocyclone configurations for Chinese hamster ovary (CHO) cell separation was investigated following an experimental design. It was shown that cell separation efficiencies for cultures of the wild-type CHO.K1 cell line and of a recombinant CHO cell line producing granulocyte-macrophage colony stimulating factor (GM-CSF) were kept above 97%. Low viability losses were observed, as measured by trypan blue exclusion and by determination of intracellular lactate dehydrogenase (LDH) released to the culture medium. Mathematical models were proposed to predict the flow rate, flow ratio and separation efficiency as a function of hydrocyclone geometry and pressure drop. When cells were monitored for any induction of apoptosis upon passage through the hydrocyclones, no increase in apoptotic cell concentration was observed within 48 h of hydrocycloning. Thus, based on the high separation efficiencies, the robustness of the equipment, and the absence of apoptosis induction, hydrocyclones seem to be specially suited for use as cell retention devices in long-term perfusion runs.     

The in vitro effects of opines and other compounds on DNAs originating from bacteria, and from healthy and tumorous plant tissues

Purified total DNAs were isolated from oncogenic or nononcogenic Agrobacterium tumefaciens cells as well as from normal and crown gall tissues. Opines (octopine, nopaline, lysopine), plant hormone (auxin IAA) and some carcinogenic compounds were used in order to correlate their effects on in vitro strand separation and synthesis of DNAs with in vivo tumorous cell multiplication. Octopine (or nopaline) induced chain opening of DNAs originating from octopine (or nopaline)-metabolizing bacteria and from same bacteria strain-induced tumorous cells. This phenomenon was measured by the increase in DNA hyperchromicity which is concentration dependent. The tested compounds stimulated the in vitro synthesis of the same DNAs. Under the same conditions, in vitro strand separation and synthesis of healthy plant DNA was not (or only slightly) enhanced, except in the case of particular hormone-connected healthy cell DNA. IAA and carcinogens stimulated in vitro synthesis and induced in vitro strand separation (dose-dependent effect) of DNAs isolated from crown gall cells and inducing bacteria. Compared to healthy cell DNAs, these DNAs were thus susceptible to structurally very diversified molecules and in this way behave as do mammalian tissue DNAs. The opine and IAA actions observed here were specific for plant tissue DNA; cancerous human or animal tissue DNAs were insensitive. By their presence in the crown gall cells, opines possibly maintain destabilized areas (required for rapid growth and division) on tumor cell DNA. The cooperative actions of IAA and opines as well as small RNA and RNA fragments on gene activation, might explain the autonomy of plant tumor cells.     

Application of microemulsion thin layer chromatography for the fingerprinting of licorice (Glycyrrhiza spp.)

Microemulsion thin layer chromatography (ME-TLC) has been developed for the fingerprinting of aqueous extract of licorice (Glycyrrhiza spp.). The separation conditions and operational processes of the method have been optimized, and its chromatographic characteristics compared with conventional TLC. The ME-TLC system is easier to operate, and with higher resolution and better reproducibility than the conventional TLC. The separation mechanism and retention behavior of ME-TLC are found to differ significantly from conventional TLC. The technique has been applied to the analysis of different licorice species including G. uralensis, G. glabra and G. inflata; and to monitor the dynamic accumulation of active ingredients in licorice plant harvested at different times during its growing cycle in a Good Agriculture Practice (GAP) research farm. Results show that without post-chromatographic derivatization, the ME-TLC fingerprinting images of different species appear as clear, well resolved bands and with strong intensities to reveal distinctively different compositional features of the samples. The technique has also been applied successfully to monitor the dynamic accumulation of active components in licorice plant as a function of growing time in an experimental licorice farm. The study demonstrates the potential of ME-TLC technique as a rapid fingerprinting tool for the authentication and quality assessment of licorice as well as other herbs.     

Structure,function, and mechanism of action of Calmodulin

The structure, function, and mechanism of action of plant calmodulins have been the subject of intense study for plant scientists during the past several years. While precedents in animal biochemistry and physiology have suggested logical starting points for studies of how calmodulin is involved in higher plant and algal cell function, recent biochemical studies have demonstrated unique structural characteristics for this highly conserved protein. Enzym‐ological analyses have demonstrated novel functional properties and provided limited insight into the molecular mechanisms of calmodulin action. This review will summarize much of the early work, but will concern itself mainly with the methods and approaches that are used to study plant and animal calmodulins as well as models for how calmodulin may be involved in plant cell function.     

Plant cell division: ROS homeostasis is required

Accumulated evidence indicates that ROS fluctuations play a critical role in cell division. Dividing plant cells rapidly respond to them. Experimental disturbance of ROS homeostasis affects: tubulin polymerization; PPB, mitotic spindle and phragmoplast assembly; nuclear envelope dynamics; chromosome separation and movement; cell plate formation. Dividing cells mainly accumulate at prophase and delay in passing through the successive cell division stages. Notably, many dividing root cells of the rhd2 Arabidopsis thaliana mutants, lacking the RHD2/AtRBOHC protein function, displayed aberrations, comparable to those induced by low ROS levels. Some protein molecules, playing key roles in signal transduction networks inducing ROS production, participate in cell division. NADPH oxidases and their regulators PLD, PI3K and ROP-GTPases, are involved in MT polymerization and organization. Cellular ROS oscillations function as messages rapidly transmitted through MAPK pathways inducing MAP activation, thus affecting MT dynamics and organization. RNS implication in cell division is also considered.