Plant Cell Function Research Articles

MicroRNA-based RNA Interference Vector for Gene Silencing in Plants

RNA interference (RNAi) is a powerful tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target genes. In this study a microRNA 159a-based binary vector was constructed which can be used for hpRNA expression. Hairpin (hp) RNA expression cassettes carrying the gene sequences are typically constructed on binary plasmid and delivered into plant cells by Agrobacterium-mediated genetic transformation. This system allows simple insertion of 21- nt target gene sequences into microRNA backbone, to facilitate the processing of microRNA hpRNA by the endogenous machinery of host, thereby producing artificial microRNA carrying the sequence of target gene(s). The functionality of new vector system was tested by silencing viral gene in transgenic plants. Strong down regulation of viral gene was observed in virus infected tobacco plants transformed with pAmiR159 vector. The processing of amIRNA leading to viral-specific sIRNA was confirmed by northern blotting. This vector system provides an important addition to the plant molecular biologists’ toolbox, which will significantly facilitate the use of RNAi technology for analyses of various gene functions in plant cells.

Comparison of the characteristics of cellulose microfibril aggregates isolated from fiber and parenchyma cells of Moso bamboo (Phyllostachys pubescens)

This study examined the relationship between the functions of plant cells and the characteristics of cellulose microfibril aggregates in the cell walls. For this purpose, the mature bamboo (Phyllostachys pubescens) culm was separated into fiber and parenchyma cells, and then the morphological and physical properties of the cellulose microfibril aggregates isolated from both cells were compared. SEM observations revealed that both fiber and parenchyma cells consist of similar microfibril aggregates approximately 15–20 nm in width. Moreover, X-ray analysis and the tensile tests of the sheets prepared from the microfibril aggregates showed that the cellulose microfibrils isolated from fiber and parenchyma cells had almost the same cellulose crystallinity and longitudinal Young’s modulus in the dry state. These results suggest that all the cellulose microfibrils synthesized in the same individual exhibit the same characteristics in the dry state regardless of cell function.

Impact of RNA Virus Infection on Plant Cell Function and Evolution

Viruses are obligate symbionts that tightly interact with their hosts to complete their life cycle. Each infected cell is confronted with the accumulation of viral products and activities that have evolved to support the replication and spread of the virus in the context of host cell functions and defense responses. Tobacco mosaic virus encodes replicase proteins and coat protein, to replicate and protect the RNA genome, and a movement protein (MP) that binds viral RNA and manipulates the size exclusion limit of plasmodesmata to facilitate the spread of the viral genomic RNA (vRNA). The MP and replicase also interfere with the cellular RNA silencing machinery that influences plant gene expression and development. Moreover, virus-infected cells stimulate the production of a systemic signal ahead of the virus front that triggers genomic recombination leading to heritable genetic changes. Thus viruses can interact with their hosts through diverse molecular interactions. Given the high mutation rate of viruses, these interactions have implications for evolutionary processes and adaptations at the virus-host interface that may contribute to eukaryotic evolution.

Novel aspects of COP9 signalosome functions revealed through analysis of hypomorphic csn mutants

The COP9 signalosome (CSN) is a conserved eukaryotic protein complex implicated in the regulation of cullin-RING type E3 ubiquitin ligases by cleaving the small peptide RUB/Nedd8 from cullins. However, detailed analysis of CSN physiological functions in Arabidopsis has been hampered by the early seedling-lethality of csn null mutants. We and others have now identified a number of viable hypomorphic csn mutants which start to reveal novel CSN-dependent activities in adult Arabidopsis plants.1 Here, we present a detailed comparative analysis of the csn5a-1 and csn2-5 mutants as a mean to improve understanding of CSN functions in plant cells. Our observations point to CSN-independent activities of CSN5 and suggest a role of the CSN in cytoskeleton assembly/organization.

Arabidopsis mitogen‐activated protein kinase MPK18 mediates cortical microtubule functions in plant cells

Mitogen-activated protein kinase (MAPK) signalling networks are important regulators of environmental responses and developmental processes in plants. To understand the role of MAPK signalling modules in the regulation of plant microtubule functions, we searched for MAPKs that interact with the dual-specificity MAPK phosphatase, PROPYZAMIDE HYPERSENSITIVE 1 (PHS1), whose mutation has previously been reported to confer hypersensitivity to microtubule-disrupting drugs in Arabidopsis. Yeast two-hybrid assays demonstrated that PHS1 specifically interacts with two MAPKs, MPK12 and MPK18. Bimolecular fluorescence complementation (BiFC) studies confirmed that the PHS1 and MPK18 proteins are physically coupled, and that this interaction occurs in the cytoplasm. At the biochemical level, in vitro dephosphorylation assays indicated that phospho-MPK18 can be dephosphorylated by recombinant PHS1. Mutant mpk18 seedlings show defects in microtubule-related functions, and have moderately stabilized microtubules. Absence of MPK18 in the phs1-1 background partially complements the phs1-1 root growth phenotypes, providing genetic evidence for involvement of MPK18 signalling in microtubule-related functions. We propose a model whereby the PHS1-MPK18 signalling module is involved in a phosphorylation/dephosphorylation switch that regulates cortical microtubule functions.

Beauvericin Decreases Cell Viability of Wheat

Recently, beauvericin (BEA) has been recognized as an important toxic compound synthesized by several Fusarium strains, infecting maize, wheat, and rice, worldwide. The effects of BEA on mammalian cells have been studied; however, its effects on the function of host plant cells are largely unknown. The purpose of our work was to assess whether BEA can affect the root and leaf cells of wheat cultivar (cv.) 'Arina' seedlings, using a cytotoxicity assay and fluorescence microscopy. Toxigenicity during wheat germination was higher in BEA-treated wheat seedlings than in non-treated seedlings (control). Leaf primordial, situated at the base and the tips of treated leaves, were more affected by BEA compared to the control when assayed in medium for cell viability measured by luminescent equipment. BEA-Treated plant cells secrete adenosine triphosphate (ATP) to the extracellular matrix and invoke more luminescence by luciferase than the non-treated seedlings. Our results were confirmed by fluorescence microscopy following '4',6-diamidino-2-phenylindole' (DAPI) staining and by confocal microscopy. In addition, the bioluminescent protein luciferase was observed in the intracellular space indicating presence of ATP. The incidence of nuclear fragmentation increased significantly in cells of seedlings treated with BEA at 40 microM concentration implying that the intracellular phytotoxin BEA plays an important role, possibly as a mediator in cell-death signalling.

Analysis of genetic factors that control shoot mineral concentrations in rapeseed (Brassica napus) in different boron environments

Mineral nutrients are essential for plant cell function, and understanding the genetic and physiological basis of mineral concentration is therefore important for the development of nutrient-efficient crop varieties that can cope with a shortage of mineral resources. In the present study, we investigated the profiles of B, Ca, Fe, Cu, Mg, P and Zn concentrations in shoots and analyzed the genetic variation in a rapeseed (Brassica napus) double haploid population at normal and deficient boron (B) levels in hydroponic conditions. Significant correlations between the concentrations of different minerals, such as Ca and Mg, Ca and P, and Cu and Fe, existed in both B environments. A total of 35 quantitative trait loci (QTL) and 74 epistatic interaction pairs for mineral concentrations were identified by whole genome analysis of QTL and epistatic interactions. The individual phenotypic contributions of the QTL ranged from 4.4% to 19.0%, and the total percentage of genetic variance that was due to QTL and epistatic interactions varied from 10.4% to 82.4%. Most of these QTL corresponded specifically to one of the two B conditions except for one stable main-effect P-QTL across the B environments. Three QTL for Ca and Mg were found to co-localize under normal B condition. These results revealed that genetic factors control mineral homeostasis in plants and multigenes involving ion transport are required to regulate mineral balance in plants under conditions of diverse nutrient stress. In addition, 26 genes involved in ion uptake and transport in Arabidopsis thaliana were in silico mapped onto the QTL intervals of B. napus by comparative genomic analysis. These candidate orthologous genes in B. napus allowed the selection of genes involved in the controlling mineral concentration that may account for the identified QTL.

Conserved Cys residue influences catalytic properties of potato endo-(1-->3)-beta-glucanase GLUB20-2.

The synthesis and degradation of (1-->3)-beta-glycosidic bonds between glucose moieties are essential metabolic processes in plant cell architecture and function. We have found that a unique, conserved cysteine residue, positioned outside the catalytic centre of potato endo-(1-->3)-beta-glucanase - product of the gluB20-2 gene, participates in determining the substrate specificity of the enzyme. The same residue is largely responsible for endo-(1-->3)-beta-glucanase inhibition by mercury ions. Our results confirm that the spatial adjustment between an enzyme and its substrate is one of the essential factors contributing to the specificity and accuracy of enzymatic reactions.

Xanthone biosynthesis in Hypericum perforatum cells provides antioxidant and antimicrobial protection upon biotic stress

Xanthone production in Hypericum perforatum (HP) suspension cultures in response to elicitation by Agrobacterium tumefaciens co-cultivation has been studied. RNA blot analyses of HP cells co-cultivated with A. tumefaciens have shown a rapid up-regulation of genes encoding important enzymes of the general phenylpropanoid pathway (PAL, phenylalanine ammonia lyase and 4CL, 4-coumarate:CoA ligase) and xanthone biosynthesis (BPS, benzophenone synthase). Analyses of HPLC chromatograms of methanolic extracts of control and elicited cells (HP cells that were co-cultivated for 24 h with A. tumefaciens) have revealed a 12-fold increase in total xanthone concentration and also the emergence of many xanthones after elicitation. Methanolic extract of elicited cells exhibited significantly higher antioxidant and antimicrobial competence than the equivalent extract of control HP cells indicating that these properties have been significantly increased in HP cells after elicitation. Four major de novo synthesized xanthones have been identified as 1,3,6,7-tetrahydroxy-8-prenyl xanthone, 1,3,6,7-tetrahydroxy-2-prenyl xanthone, 1,3,7-trihydroxy-6-methoxy-8-prenyl xanthone and paxanthone. Antioxidant and antimicrobial characterization of these de novo xanthones have revealed that xanthones play dual function in plant cells during biotic stress: (1) as antioxidants to protect the cells from oxidative damage and (2) as phytoalexins to impair the pathogen growth.

Seed Germination and Seedling Growth under Simulated Microgravity Causes Alterations in Plant Cell Proliferation and Ribosome Biogenesis

The study of the modifications induced by altered gravity in functions of plant cells is a valuable tool for the objective of the survival of terrestrial organisms in conditions different from those of the Earth. We have used the system “cell proliferation–ribosome biogenesis”, two inter-related essential cellular processes, with the purpose of studying these modifications. Arabidopsis seedlings belonging to a transformed line containing the reporter gene GUS under the control of the promoter of the cyclin gene CYCB1, a cell cycle regulator, were grown in a Random Positioning Machine, a device known to accurately simulate microgravity. Samples were taken at 2, 4 and 8 days after germination and subjected to biometrical analysis and cellular morphometrical, ultrastructural and immunocytochemical studies in order to know the rates of cell proliferation and ribosome biogenesis, plus the estimation of the expression of the cyclin gene, as an indication of the state of cell cycle regulation. Our results show that cells divide more in simulated microgravity in a Random Positioning Machine than in control gravity, but the cell cycle appears significantly altered as early as 2 days after germination. Furthermore, higher proliferation is not accompanied by an increase in ribosome synthesis, as is the rule on Earth, but the functional markers of this process appear depleted in simulated microgravity-grown samples. Therefore, the alteration of the gravitational environmental conditions results in a considerable stress for plant cells, including those not specialized in gravity perception.

The plant-specific TFIIB-related protein, pBrp, is a general transcription factor for RNA polymerase I

TFIIB and BRF are general transcription factors (GTFs) for eukaryotic RNA polymerases II and III, respectively, and have important functions in transcriptional initiation. In this study, the third type of TFIIB-related protein, pBrp, found in plant lineages was characterized in the red alga Cyanidioschyzon merolae. Chromatin immunoprecipitation analysis revealed that CmpBrp specifically occupied the rDNA promoter region in vivo, and the occupancy was proportional to de novo 18S rRNA synthesis. Consistently, CmpBrp and CmTBP cooperatively bound the rDNA promoter region in vitro, and the binding site was identified at a proximal downstream region of the transcription start point. alpha-Amanitin-resistant transcription from the rDNA promoter in crude cell lysate was severely inhibited by the CmpBrp antibody and was also inhibited when DNA template with a mutated CmpBrp-CmTBP binding site was used. CmpBrp was shown to co-immunoprecipitate and co-localize with the RNA polymerase I subunit, CmRPA190, in the cell. Thus, together with comparative studies of Arabidopsis pBrp, we concluded that pBrp is a GTF for RNA polymerase I in plant cells.

Proteomics applied on plant abiotic stresses: Role of heat shock proteins (HSP)

The most crucial function of plant cell is to respond against stress induced for self-defence. This defence is brought about by alteration in the pattern of gene expression: qualitative and quantitative changes in proteins are the result, leading to modulation of certain metabolic and defensive pathways. Abiotic stresses usually cause protein dysfunction. They have an ability to alter the levels of a number of proteins which may be soluble or structural in nature. Nowadays, in higher plants high-throughput protein identification has been made possible along with improved protein extraction, purification protocols and the development of genomic sequence databases for peptide mass matches. Thus, recent proteome analysis performed in the vegetal Kingdom has provided new dimensions to assess the changes in protein types and their expression levels under abiotic stress. As reported in this review, specific and novel proteins, protein-protein interactions and post-translational modifications have been identified, which play a role in signal transduction, anti-oxidative defence, anti-freezing, heat shock, metal binding etc. However, beside specific proteins production, plants respond to various stresses in a similar manner by producing heat shock proteins (HSPs), indicating a similarity in the plant's adaptive mechanisms; in plants, more than in animals, HSPs protect cells against many stresses. A relationship between ROS and HSP also seems to exist, corroborating the hypothesis that during the course of evolution, plants were able to achieve a high degree of control over ROS toxicity and are now using ROS as signalling molecules to induce HSPs.

Antisense oligodeoxynucleotide inhibition as a potent diagnostic tool for gene function in plant biology

Antisense oligodeoxynucleotide (ODN) inhibition emerges as an effective means for probing gene function in plant cells. Employing this method we have established the importance of the SUSIBA2 transcription factor for regulation of starch synthesis in barley endosperm, and arrived at a model for the role of the SUSIBAs in sugar signaling and source-sink commutation during cereal endosperm development. In this addendum we provide additional data demonstrating the suitability of the antisense ODN technology in studies on starch branching enzyme activities in barley leaves. We also comment on the mechanism for ODN uptake in plant cells.

Attacking the defenders: plant viruses fight back

Plants use RNA silencing mechanisms and produce short-interfering RNA (siRNA) molecules in a defense response against viral infection. To counter this defense response, viruses produce suppressor proteins, which can block the host silencing pathway or interfere with its function in plant cells. The targets for many viral suppressors and the mechanisms by which they function in plant cells are still largely unknown. Recent reports describe that the 2b suppressor of the Cucumber mosaic virus binds ARGONAUTE and that the P0 suppressor of Polerovirus targets ARGONAUTE to degradation. Another report has revealed that the V2 suppressor of tomato yellow mosaic virus binds the coiled-coil protein suppressor of the gene-silencing SGS3 homolog. These reports provide novel insight into the mechanisms developed by viruses to disable the defense system of the plant.

PSAT RNA Interference Vectors: A Modular Series for Multiple Gene Down-Regulation in Plants

RNA interference (RNAi) is a powerful tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target genes, enabling loss-of-function studies in living cells. Hairpin (hp) RNA expression cassettes are typically constructed on binary plasmids and delivered into plant cells by Agrobacterium-mediated genetic transformation. Realizing the importance of RNAi for basic plant research, various vectors have been developed for RNAi-mediated gene silencing, allowing the silencing of single target genes in plant cells. To further expand the collection of available tools for functional genomics in plant species, we constructed a set of modular vectors suitable for hpRNA expression under various constitutive promoters. Our system allows simple cloning of the target gene sequences into two distinct multicloning sites and its modular design provides a straightforward route for replacement of the expression cassette's regulatory elements. More importantly, our system was designed to facilitate the assembly of several hpRNA expression cassettes on a single plasmid, thereby enabling the simultaneous suppression of several target genes from a single vector. We tested the functionality of our new vector system by silencing overexpressed marker genes (green fluorescent protein, DsRed2, and nptII) in transgenic plants. Various combinations of hpRNA expression cassettes were assembled in binary plasmids; all showed strong down-regulation of the reporter genes in transgenic plants. Furthermore, assembly of all three hpRNA expression cassettes, combined with a fourth cassette for the expression of a selectable marker, resulted in down-regulation of all three different marker genes in transgenic plants. This vector system provides an important addition to the plant molecular biologist's toolbox, which will significantly facilitate the use of RNAi technology for analyses of multiple gene function in plant cells.

Root-knot nematodes manipulate plant cell functions during a compatible interaction

Sedentary endoparasitic nematodes are root parasites that interact with their hosts in a remarkable way. These obligate biotrophic pathogens establish an intimate relationship with their host plants, inducing the redifferentiation of root cells into specialized feeding cells. The successful establishment of feeding cells is essential for nematode development. Root-knot nematodes, of the genus Meloidogyne, have evolved strategies enabling them to induce feeding cell formation in thousands of plant species, probably by manipulating fundamental elements of plant cell development. Feeding cells enlarge and are converted into multinucleate giant cells through synchronous nuclear divisions without cell division. Fully differentiated giant cells may contain more than a hundred polyploid nuclei that may have undergone extensive endoreduplication. Hyperplasia and hypertrophy of the surrounding cells lead to the formation of the typical root gall. Giant cell formation requires extensive changes to gene expression. The induction of feeding cells remains poorly understood, but it is thought that effectors secreted by the nematode play a key role in parasitism, with potential direct effects on recipient host cells. In this review, we focus on the most recent investigations of the molecular basis of the plant-root-knot nematode interaction. Recently, microarray technology has been used to study the plant response to Meloidogyne spp. infection. Such a genome-wide expression profiling provides a global view of transcriptional changes, especially for genes involved in cell wall, transport processes and plant defense responses during giant cell formation. The identification of nematode-responsive plant genes constitutes a major step toward understanding how root-knot nematodes dramatically alter root development to induce and maintain giant cells. The characterization of nematode secretions as parasitism effectors and the development of RNAi technology should improve our understanding of the molecular events and regulatory mechanisms involved in plant parasitism. Finally, Meloidogyne genome sequences should provide further insight into plant-root-knot nematode interactions.

Proteomics-based dissection of stress-responsive pathways in plants

Abiotic stress has an ability to alter the levels of a number of proteins, which may be soluble or structural in nature or which may exist before and after folding in the plant cell. The most crucial function of plant cell is to respond to stress by developing defence mechanisms. This defence is brought about by alteration in the pattern of gene expression. This leads to modulation of certain metabolic and defensive pathways. Owing to gene expression altered under stress, qualitative and quantitative changes in proteins are obvious. These proteins might play a role in signal transduction, antioxidative defence, antifreezing, heat shock, metal binding, antipathogenesis or osmolyte synthesis. A significant part of the literature shows the quantitative and qualitative changes in proteins, mainly employing western analysis, enzymatic kinetics, fraction isolation, one-dimensional SDS-PAGE electrophoresis, etc. Fortunately, recent developments in sensitivity and accuracy for proteome analysis have provided new dimensions to assess the changes in protein types and their expression levels under stress. The novel aim of this review is to do a side-by-side comparison of the proteins that are induced or overexpressed under abiotic stress, examining those from biochemical literature and the ones observed, sequenced and identified using the advanced proteomics and bioinformatic techniques.

A gene from the cellulose synthase-like C family encodes a β-1,4 glucan synthase

Despite the central role of xyloglucan (XyG) in plant cell wall structure and function, important details of its biosynthesis are not understood. To identify the gene(s) responsible for synthesizing the beta-1,4 glucan backbone of XyG, we exploited a property of nasturtium (Tropaeolum majus) seed development. During the last stages of nasturtium seed maturation, a large amount of XyG is deposited as a reserve polysaccharide. A cDNA library was produced from mRNA isolated during the deposition of XyG, and partial sequences of 10,000 cDNA clones were determined. A single member of the C subfamily from the large family of cellulose synthase-like (CSL) genes was found to be overrepresented in the cDNA library. Heterologous expression of this gene in the yeast Pichia pastoris resulted in the production of a beta-1,4 glucan, confirming that the CSLC protein has glucan synthase activity. The Arabidopsis CSLC4 gene, which is the gene with the highest sequence similarity to the nasturtium CSL gene, is coordinately expressed with other genes involved in XyG biosynthesis. These and other observations provide a compelling case that the CSLC gene family encode proteins that synthesize the XyG backbone.

The sliding theory of cytoplasmic streaming: fifty years of progress

Fifty years ago, an important paper appeared in Botanical Magazine Tokyo. Kamiya and Kuroda proposed a sliding theory for the mechanism of cytoplasmic streaming. This pioneering study laid the basis for elucidation of the molecular mechanism of cytoplasmic streaming--the motive force is generated by the sliding of myosin XI associated with organelles along actin filaments, using the hydrolysis energy of ATP. The role of the actin-myosin system in various plant cell functions is becoming evident. The present article reviews progress in studies on cytoplasmic streaming over the past 50 years.

The miRNA156/157 recognition element in the 3′ UTR of the Arabidopsis SBP box gene SPL3 prevents early flowering by translational inhibition in seedlings

miRNAs are a class of versatile small RNAs that control gene expression post-transcriptionally, governing many facets of plant cell functions. They interact with their target mRNA at a site of sequence complementarity and modulate their expression levels. Here, we provide evidence, based on transient assays and stable transgenic lines, that the 3' UTR of the Arabidopsis SBP box gene SPL3 contains a functional miRNA-responsive element (MRE) that is complementary to miR156 and miRNA157. Seedlings of transgenic lines constitutively over-expressing an SPL3 transgene either carrying an unaltered or a disrupted MRE accumulate considerable levels of SPL3 transcripts. However, while the unaltered MRE UTR does not allow the expression of detectable levels of SPL3 protein, the altered MRE does. Translational inhibition thus provides an important mechanism for miRNA-mediated post-transcriptional repression of SPL3. As a consequence of precocious translation of the constitutively expressed SPL3 transgene, due to the absence of a functional MRE, plants exhibit very early flowering in addition to frequent morphological changes.