Determination of in vivo disulfide-bonded proteins in Arabidopsis

Protein thiol-disulfide oxidoreduction plays an important role in redox regulation of cellular processes. Here we present a proteomic approach to visualize and map in vivo disulfide-bonded proteins in plants. A proteomic map of the disulfide-bonded proteins was achieved using 2D gel electrophoresis of Arabidopsis protein extract. Along with novel proteins identified as potentially redox regulated, we have also shown the feasibility of mapping some of the cysteines involved in the formation of disulfide bonds. This study presents an important tool for characterizing redox-regulated proteins.     

Saturating the genetic map of Arabidopsis thaliana with embryonic mutations

One goal of the Arabidopsis genome project is to identify every gene with an essential function in growth and development. Towards that end, the results are reported here of a mapping project designed to enhance the linkage map of Arabidopsis and establish a valuable resource of mutations in essential genes with known map locations. Embryo-defective (emb) mutations were chosen because they represent the most common heritable defect identified following mutagenesis in Arabidopsis. Multiple marker lines with easily scored phenotypes were constructed to facilitate mapping efforts. Recombination data were obtained for 169 mutants defective in embryo-genesis. The chromosomal locations of 110 emb genes are presented in this report. Twenty-six of these genes are tagged with T-DNA. Nine other mutants isolated following seed transformation appear to contain chromosomal translocations. Another 31 mutant genes in the collectiohave been assigned to a linkage group but not yet placed on the map. Nineteen examples of duplicate alleles have also been found. This is consistent with the estimate that approximately 500 genes readily mutate to give an embryo-defective phenotype in Arabidopsis. With continued progress, it may therefore be possible to approach saturation for this important class of mutations. Molecular cloning of these genes should be facilitated by identifying cDNAs and genomic sequences that map to similar locations.     

Protein Interaction Network of Arabidopsis thaliana Female Gametophyte Development Identifies Novel Proteins and Relations

Although the female gametophyte in angiosperms consists of just seven cells, it has a complex biological network. In this study, female gametophyte microarray data from Arabidopsis thaliana were integrated into the Arabidopsis interactome database to generate a putative interaction map of the female gametophyte development including proteome map based on biological processes and molecular functions of proteins. Biological and functional groups as well as topological characteristics of the network were investigated by analyzing phytohormones, plant defense, cell death, transporters, regulatory factors, and hydrolases. This approach led to the prediction of critical members and bottlenecks of the network. Seventy-four and 24 upregulated genes as well as 171 and 3 downregulated genes were identified in subtracted networks based on biological processes and molecular function respectively, including novel genes such as the pathogenesis-related protein 4, ER type Ca²⁺ ATPase 3, dihydroflavonol reductase, and ATP disulfate isomerase. Biologically important relationships between genes, critical nodes, and new essential proteins such as AT1G26830, AT5G20850, CYP74A, AT1G42396, PR4 and MEA were found in the interactome''s network. The positions of novel genes, both upregulated and downregulated, and their relationships with biological pathways, in particular phytohormones, were highlighted in this study.     

The essential role of the phosphorylated pathway of serine biosynthesis in Arabidopsis

In plants, 3 different pathways of serine biosynthesis have been described: the Glycolate pathway, which is associated with photorespiration, and 2 non-photorespiratory pathways, the Glycerate and the Phosphorylated pathways. The Phosphorylated Pathway of Serine Biosynthesis (PPSB) has been known since the 1950s, but has been studied relatively little, probably because it was considered of minor significance as compared with the Glycolate pathway. In the associated study¹, we described for the first time in plants the in vivo functional characterization of the PPSB, by targeting the phosphoserine phosphatase (PSP1), the last enzyme of the pathway. Following a gain- and loss-of-function approach in Arabidopsis, we provided genetic and molecular evidence for the essential role of PSP1 for embryo and pollen development, and for proper root growth. A metabolomics study indicated that the PPSB affects glycolysis, the Krebs cycle, and the biosynthesis of several amino acids, which suggests that this pathway is an important link connecting metabolism and development. The mechanisms underlying the essential functions of PSP1 are discussed.     

Ectopic overexpression of the aluminum-induced protein gene from Panax ginseng enhances heavy metal tolerance in transgenic Arabidopsis

Aluminum (Al), one of the most abundant metals in agricultural soils, significantly limits crop growth and productivity in acidic soil by inhibiting root elongation. Al ions, especially Al³⁺, have a toxic effect on both plant and animal cells under low-pH conditions. We first isolated and characterized aluminum-induced protein (AIP) cDNA from a 4-year-old root of Panax ginseng Meyer. This cDNA encodes an open reading frame of 711 bp with a deduced amino acid sequence of 236 residues. The calculated molecular mass of the mature protein is approximately 58.9 kDa with a predicated isoelectric point of 5.13. The Panax ginseng AIP (PgAIP) contains a domain also present in wheat aluminum-induced protein 7 (Wali7) and shares homology with the AIPs of other species, including Codonopis and Arabidopsis. The PgAIP gene was abundantly expressed in the plant’s leaves and was up-regulated by Al exposure. The functional role of PgAIP in Al tolerance was further validated through its overexpression in Arabidopsis. Transgenic Arabidopsis plants overexpressing the PgAIP gene showed enhanced Al tolerance in terms of root growth when compared to wild-type plants, suggesting PgAIP is important in plant defense against Al toxicity. Confocal analysis of CFP-tagging PgAIP in Arabidopsis showed subcellular localization in the plasma membrane. Our results suggest that PgAIP in the plasma membrane plays an important role in the protection of plant cells against heavy metal exposure.     

A Theoretical Model for ROP Localisation by Auxin in Arabidopsis Root Hair Cells

Background

Local activation of Rho GTPases is important for many functions including cell polarity, morphology, movement, and growth. Although a number of molecules affecting Rho-of-Plants small GTPase (ROP) signalling are known, it remains unclear how ROP activity becomes spatially organised. Arabidopsis root hair cells produce patches of ROP at consistent and predictable subcellular locations, where root hair growth subsequently occurs.

Methodology/Principal Findings

We present a mathematical model to show how interaction of the plant hormone auxin with ROPs could spontaneously lead to localised patches of active ROP via a Turing or Turing-like mechanism. Our results suggest that correct positioning of the ROP patch depends on the cell length, low diffusion of active ROP, a gradient in auxin concentration, and ROP levels. Our theory provides a unique explanation linking the molecular biology to the root hair phenotypes of multiple mutants and transgenic lines, including OX-ROP, CA-rop, aux1, axr3, tip1, eto1, etr1, and the triple mutant aux1 ein2 gnom ^eb.

Conclusions/Significance

We show how interactions between Rho GTPases (in this case ROPs) and regulatory molecules (in this case auxin) could produce characteristic subcellular patterning that subsequently affects cell shape. This has important implications for research on the morphogenesis of plants and other eukaryotes. Our results also illustrate how gradient-regulated Turing systems provide a particularly robust and flexible mechanism for pattern formation.     

Overexpression of a cytosol-localized rhamnose biosynthesis protein encoded by Arabidopsis RHM1 gene increases rhamnose content in cell wall

l-Rhamnose (Rha) is an important constituent of pectic polysaccharides, a major component of the cell walls of Arabidopsis, which is synthesized by three enzymes encoded by AtRHM1, AtRHM2/AtMUM4, and AtRHM3. Despite the finding that RHM1 is involved in root hair formation in Arabidopsis, experimental evidence is still lacking for the in vivo enzymatic activity and subcellular compartmentation of AtRHM1 protein. AtRHM1 displays high similarity to the other members of RHM family in Arabidopsis and in other plant species such as rice and grape. Expression studies with AtRHM1 promoter-GUS fusion gene showed that AtRHM1 was expressed almost ubiquitously, with stronger expression in roots and cotyledons of young seedlings and inflorescences. GFP::AtRHM1 fusion protein was found to be localized in the cytosol of cotyledon cells and of petiole cells of cotyledon, indicating that AtRHM1 is a cytosol-localized protein. The overexpression of AtRHM1 gene in Arabidopsis resulted in an increase of rhamnose content as much as 40% in the leaf cell wall compared to the wild type as well as an alteration in the contents of galactose and glucose. Fourier-transform infrared analyses revealed that surplus rhamnose upon AtRHM1 overexpression contributes to the construction of rhamnogalacturonan.     

Learning Probabilistic Features for Robotic Navigation Using Laser Sensors

SLAM is a popular task used by robots and autonomous vehicles to build a map of an unknown environment and, at the same time, to determine their location within the map. This paper describes a SLAM-based, probabilistic robotic system able to learn the essential features of different parts of its environment. Some previous SLAM implementations had computational complexities ranging from O(Nlog(N)) to O(N²), where N is the number of map features. Unlike these methods, our approach reduces the computational complexity to O(N) by using a model to fuse the information from the sensors after applying the Bayesian paradigm. Once the training process is completed, the robot identifies and locates those areas that potentially match the sections that have been previously learned. After the training, the robot navigates and extracts a three-dimensional map of the environment using a single laser sensor. Thus, it perceives different sections of its world. In addition, in order to make our system able to be used in a low-cost robot, low-complexity algorithms that can be easily implemented on embedded processors or microcontrollers are used.     

How to map ses, a mutant of Arabidopsis thaliana affecting pollen development

In Arabidopsis, map-based cloning has been developed to an effective method in mutant genetic analysis because high-density markers are available, candidate genes or genomic sequences can be amplified by PCR, and transgenic techniques are simplified. Mutant ses named from shortened early-stage siliques was used as an example to show how to map a mutant in this way. By the process of bulked segregants analysis, linkage testing, large-scale and fine-scale mapping, mutant ses was narrowed into a 67 kb interval from CER448792 (2000541 bp) to CER464544 (2067844 bp) crossing over the right of BAC F12K11 to the left of the BAC F4H5 including at most 22 putative genes on the top of chromosome 1. In sequence-based map of Arabidopsis genes with mutant phenotype (SMAGMP) mutant ses was between AT1g06150 (EMB1444) and AT1g08060 (MOM). The ses mapping also showed that developed markers on polymorphism site of CAPC not only were simplified but worked well. Twenty-four markers from CAPC used in the mapping maybe help Arabidopsis researchs with others and the methods related to ses mapping also gave an example of positional cloning. The text was submitted by the authors in English.     

Overexpression of glucosyltransferase UGT85A1 influences trans-zeatin homeostasis and trans-zeatin responses likely through O-glucosylation

Trans-zeatin is a kind of cytokinins that plays a crucial role in plant growth and development. The master trans-zeatin O-glucosyltransferase of Arabidopsis thaliana, UGT85A1, has been previously identified through biochemical approach. To determine the in planta role of UGT85A1 gene, the characterization of transgenic Arabidopsis plants overexpressing UGT85A1 was carried out. Under normal conditions, transgenic Arabidopsis did not display clearly altered phenotypes. A remarkable alteration is that the accumulation level of the trans-zeatin O-glucosides was significantly increased in UGT85A1 overexpressing transgenic Arabidopsis, while other forms of cytokinins kept the similar concentrations compared to the wild type. When treated with exogenously applied trans-zeatin, UGT85A1 overexpressing Arabidopsis showed much less sensitivity to trans-zeatin in primary root elongation and lateral root formation. Meanwhile, the chlorophyll content of detached leaves of transgenic Arabidopsis was much lower than wild type. Studies of spatial–temporal expression patterns showed that UGT85A1 was mainly expressed in the early seedlings and developing seeds. Analysis of subcellular localization suggested that UGT85A1 was localized to cytoplasm and nucleus. Taken together, our data suggest that overexpression of Arabidopsis glucosyltransferase UGT85A1 influences trans-zeatin homeostasis and trans-zeatin responses likely through O-glucosylation in planta.     

Generation of transgenic Arabidopsis plants expressing mcherry-fused organelle marker proteins

In eukaryotic cells, a major proportion of the cellular proteins localize to various subcellular organelles where they are involved in organelle-specific cellular processes. Thus, the localization of a particular protein in the cell is an important part of understanding the physiological role of the protein in the cell. Various approaches such as subcellular fractionation, immunolocalization and live imaging have been used to define the localization of organellar proteins. Of these various approaches, the most powerful one is the live imaging because it can show in vivo dynamics of protein localization depending on cellular and environmental conditions without disturbing cellular structures. However, the live imaging requires the ability to detect the organelles in live cells. In this study, we report generation of a new set of transgenic Arabidopsis plants using various organelle marker proteins fused to a fluorescence protein, monomeric Cherry (mCherry). All these markers representing different subcellular organelles such as chloroplasts, mitochondria, peroxisomes, endoplasmic reticulum (ER) and lytic vacuole showed clear and specific signals regardless of the cell types and tissues. These marker lines can be used to determine localization of organellar proteins by colocalization and also to study the dynamics of organelles under various developmental and environmental conditions.     

Enhancement of seed germination in high salinity by engineering mannitol expression in Arabidopsis thaliana

The bacterial gene mtlD, which encodes mannitol 1-phosphate dehydrogenase (E. C. 1. 1. 1. 17), was transformed into Arabidopsis thaliana and expressed under control of the CaMV 35S promoter. MtlD-transformants accumulated mannitol, a sugar alcohol that is not normally found in Arabidopsis. Amounts of soluble carbohydrates, sucrose, glucose, fructose, myo-inositol and mannitol were determined in different tissues of wild-type and transgenic plants. We estimated that less than 1& of the carbon assimilated was converted into mannitol by the transgenic plants. The establishment of individual transformed lines (after self-crossing three times) resulted in high and low mannitol-producing lines which were stably maintained. The presence of mannitol did not alter plant appearance or growth habit. When MtlD-expressing seeds and control seeds (T3 generation) were imbibed with solutions containing NaCl (range 0 to 400 mol m^?3), transgenic seeds containing mannitol germinated in medium supplemented with up to 400 mol m^?3 NaCl, while control seeds ceased germination at 100 mol m^?3 NaCl. It is doubtful whether the ability to germinate in high salt was a result of an osmotic effect exerted by elevated levels of mannitol, considering that mannitol concentrations were in the mol m^?3 range in seeds. A specific effect of polyols, for example on the integrity of subcellular membranes or enzymes, cannot be excluded.     

Arabidopsis Calmodulin-binding Protein IQ67-Domain 1 Localizes to Microtubules and Interacts with Kinesin Light Chain-related Protein-1

Calcium (Ca²⁺) is a key second messenger in eukaryotes and regulates diverse cellular processes, most notably via calmodulin (CaM). In Arabidopsis thaliana, IQD1 (IQ67 domain 1) is the founding member of the IQD family of putative CaM targets. The 33 predicted IQD proteins share a conserved domain of 67 amino acids that is characterized by a unique arrangement of multiple CaM recruitment motifs, including so-called IQ motifs. Whereas IQD1 has been implicated in the regulation of defense metabolism, the biochemical functions of IQD proteins remain to be elucidated. In this study we show that IQD1 binds to multiple Arabidopsis CaM and CaM-like (CML) proteins in vitro and in yeast two-hybrid interaction assays. CaM overlay assays revealed moderate affinity of IQD1 to CaM2 (K_d ∼ 0.6 μm). Deletion mapping of IQD1 demonstrated the importance of the IQ67 domain for CaM2 binding in vitro, which is corroborated by interaction of the shortest IQD member, IQD20, with Arabidopsis CaM/CMLs in yeast. A genetic screen of a cDNA library identified Arabidopsis kinesin light chain-related protein-1 (KLCR1) as an IQD1 interactor. The subcellular localization of GFP-tagged IQD1 proteins to microtubules and the cell nucleus in transiently and stably transformed plant tissues (tobacco leaves and Arabidopsis seedlings) suggests direct interaction of IQD1 and KLCR1 in planta that is supported by GFP∼IQD1-dependent recruitment of RFP∼KLCR1 and RFP∼CaM2 to microtubules. Collectively, the prospect arises that IQD1 and related proteins provide Ca²⁺/CaM-regulated scaffolds for facilitating cellular transport of specific cargo along microtubular tracks via kinesin motor proteins.     

RNA-Seq Analysis of the Response of the Halophyte,Mesembryanthemum crystallinum (Ice Plant) to High Salinity

Understanding the molecular mechanisms that convey salt tolerance in plants is a crucial issue for increasing crop yield. The ice plant (Mesembryanthemum crystallinum) is a halophyte that is capable of growing under high salt conditions. For example, the roots of ice plant seedlings continue to grow in 140 mM NaCl, a salt concentration that completely inhibits Arabidopsis thaliana root growth. Identifying the molecular mechanisms responsible for this high level of salt tolerance in a halophyte has the potential of revealing tolerance mechanisms that have been evolutionarily successful. In the present study, deep sequencing (RNAseq) was used to examine gene expression in ice plant roots treated with various concentrations of NaCl. Sequencing resulted in the identification of 53,516 contigs, 10,818 of which were orthologs of Arabidopsis genes. In addition to the expression analysis, a web-based ice plant database was constructed that allows broad public access to the data. The results obtained from an analysis of the RNAseq data were confirmed by RT-qPCR. Novel patterns of gene expression in response to high salinity within 24 hours were identified in the ice plant when the RNAseq data from the ice plant was compared to gene expression data obtained from Arabidopsis plants exposed to high salt. Although ABA responsive genes and a sodium transporter protein (HKT1), are up-regulated and down-regulated respectively in both Arabidopsis and the ice plant; peroxidase genes exhibit opposite responses. The results of this study provide an important first step towards analyzing environmental tolerance mechanisms in a non-model organism and provide a useful dataset for predicting novel gene functions.