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1.
Int J Mol Sci ; 20(13)2019 Jul 06.
Article in English | MEDLINE | ID: mdl-31284602

ABSTRACT

The DUF642 protein family is found exclusively in spermatophytes and is represented by 10 genes in Arabidopsis and in most of the 24 plant species analyzed to date. Even though the primary structure of DUF642 proteins is highly conserved in different spermatophyte species, studies of their expression patterns in Arabidopsis have shown that the spatial-temporal expression pattern for each gene is specific and consistent with the phenotypes of the mutant plants studied so far. Additionally, the regulation of DUF642 gene expression by hormones and environmental stimuli was specific for each gene, showing both up- and down-regulation depending of the analyzed tissue and the intensity or duration of the stimuli. These expression patterns suggest that the DUF642 genes are involved throughout the development and growth of plants. In general, changes in the expression patterns of DUF642 genes can be related to changes in pectin methyl esterase activity and/or to changes in the degree of methyl-esterified homogalacturonans during plant development in different cell types. Thus, the regulation of pectin methyl esterases mediated by DUF642 genes could contribute to the regulation of the cell wall properties during plant growth.


Subject(s)
Cell Wall/metabolism , Plant Development , Plant Proteins/metabolism , Gene Expression Regulation, Plant , Genes, Plant , Plant Development/genetics , Plant Proteins/genetics
2.
Plant Cell ; 28(9): 2326-2341, 2016 Sep.
Article in English | MEDLINE | ID: mdl-27577790

ABSTRACT

Eukaryotes contain three types of lipid kinases that belong to the phosphatidylinositol 3-kinase (PI3K) family. In plants and Saccharomyces cerevisiae, only PI3K class III family members have been identified. These enzymes regulate the innate immune response, intracellular trafficking, autophagy, and senescence. Here, we report that RNAi-mediated downregulation of common bean (Phaseolus vulgaris) PI3K severely impaired symbiosis in composite P. vulgaris plants with endosymbionts such as Rhizobium tropici and Rhizophagus irregularis Downregulation of Pv-PI3K was associated with a marked decrease in root hair growth and curling. Additionally, infection thread growth, root-nodule number, and symbiosome formation in root nodule cells were severely affected. Interestingly, root colonization by AM fungi and the formation of arbuscules were also abolished in PI3K loss-of-function plants. Furthermore, the transcript accumulation of genes encoding proteins known to interact with PI3K to form protein complexes involved in autophagy was drastically reduced in these transgenic roots. RNAi-mediated downregulation of one of these genes, Beclin1/Atg6, resulted in a similar phenotype as observed for transgenic roots in which Pv-PI3K had been downregulated. Our findings show that an autophagy-related process is crucial for the mutualistic interactions of P. vulgaris with beneficial microorganisms.

3.
Sensors (Basel) ; 15(1): 855-67, 2015 Jan 06.
Article in English | MEDLINE | ID: mdl-25569758

ABSTRACT

Emerging evidence indicates that some reactive oxygen species (ROS), such as the superoxide anion radical and hydrogen peroxide (H2O2), are central regulators of plant responses to biotic and abiotic stresses. Thus, the cellular levels of ROS are thought to be tightly regulated by an efficient and elaborate pro- and antioxidant system that modulates the production and scavenging of ROS. Until recently, studies of ROS in plant cells have been limited to biochemical assays and the use of fluorescent probes; however, the irreversible oxidation of these fluorescent probes makes it impossible to visualize dynamic changes in ROS levels. In this work, we describe the use of Hyper, a recently developed live cell probe for H2O2 measurements in living cells, to monitor oxidative stress in Arabidopsis roots subjected to aluminum treatment. Hyper consists of a circularly permuted YFP (cpYFP) inserted into the regulatory domain of the Escherichia coli hydrogen peroxide-binding protein (OxyR), and is a H2O2-specific ratiometric, and therefore quantitative, probe that can be expressed in plant and animal cells. Now we demonstrate that H2O2 levels drop sharply in the elongation zone of roots treated with aluminum. This response could contribute to root growth arrest and provides evidence that H2O2 is involved in early Al sensing.


Subject(s)
Aluminum/toxicity , Arabidopsis/growth & development , Biosensing Techniques , Hydrogen Peroxide/analysis , Plant Roots/growth & development , Arabidopsis/drug effects , Intracellular Space/metabolism , Plant Roots/drug effects , Plants, Genetically Modified
4.
Plant Cell Physiol ; 55(3): 580-92, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24399235

ABSTRACT

Legume plants secrete signaling molecules called flavonoids into the rhizosphere. These molecules activate the transcription of rhizobial nod genes, which encode proteins involved in the synthesis of signaling compounds named Nod factors (NFs). NFs, in turn, trigger changes in plant gene expression, cortical cell dedifferentiation and mitosis, depolarization of the root hair cell membrane potential and rearrangement of the actin cytoskeleton. Actin polymerization plays an important role in apical growth in hyphae and pollen tubes. Using sublethal concentrations of fluorescently labeled cytochalasin D (Cyt-Fl), we visualized the distribution of filamentous actin (F-actin) plus ends in living Phaseolus vulgaris and Arabidopsis root hairs during apical growth. We demonstrated that Cyt-Fl specifically labeled the newly available plus ends of actin microfilaments, which probably represent sites of polymerization. The addition of unlabeled competing cytochalasin reduced the signal, suggesting that the labeled and unlabeled forms of the drug bind to the same site on F-actin. Exposure to Rhizobium etli NFs resulted in a rapid increase in the number of F-actin plus ends in P. vulgaris root hairs and in the re-localization of F-actin plus ends to infection thread initiation sites. These data suggest that NFs promote the formation of F-actin plus ends, which results in actin cytoskeleton rearrangements that facilitate infection thread formation.


Subject(s)
Actins/metabolism , Lipopolysaccharides/pharmacology , Phaseolus/drug effects , Phaseolus/metabolism , Plant Roots/drug effects , Plant Roots/metabolism , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/metabolism
5.
Plant Signal Behav ; 9(8): e29401, 2014.
Article in English | MEDLINE | ID: mdl-25763621

ABSTRACT

In plants, the actin cytoskeleton is a prime regulator of cell polarity, growth, and cytoplasmic streaming. Tip growth, as observed in root hairs, caulonema, and pollen tubes, is governed by many factors, including calcium gradients, exocytosis and endocytosis, reactive oxygen species, and the cytoskeleton. Several studies indicate that the polymerization of G-actin into F-actin also contributes to tip growth. The structure and function of F-actin within the apical dome is variable, ranging from a dense meshwork to sparse single filaments. The presence of multiple F-actin structures in the elongating apices of tip-growing cells suggests that this cytoskeletal array is tightly regulated. We recently reported that sublethal concentrations of fluorescently labeled cytochalasin could be used to visualize the distribution of microfilament plus ends using fluorescence microscopy, and found that the tip region of the growing root hair cells of a legume plant exhibits a clear response to the nodulation factors secreted by Rhizobium. (1) In this current work, we expanded our analysis using confocal microscopy and demonstrated the existence of highly dynamic fluorescent foci along Arabidopsis root hair cells. Furthermore, we show that the strongest fluorescence signal accumulates in the tip dome of the growing root hair and seems to be in close proximity to the apical plasma membrane. Based on these findings, we propose that actin polymerization within the dome of growing root hair cells regulates polar growth.


Subject(s)
Actin Cytoskeleton , Actins/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Meristem/metabolism , Plant Roots/metabolism , Polymerization , Arabidopsis/growth & development , Hair , Meristem/growth & development , Microscopy, Confocal , Plant Roots/growth & development
6.
Methods Enzymol ; 527: 275-90, 2013.
Article in English | MEDLINE | ID: mdl-23830637

ABSTRACT

Reactive oxygen species (ROS) are highly reactive reduced oxygen molecules that play a myriad of roles in animal and plant cells. In plant cells, the production of ROS occurs as a result of aerobic metabolism during respiration and photosynthesis. Therefore mitochondria, chloroplasts, and peroxisomes constitute an important source of ROS. However, they can be produced in response to many physiological stimuli such as pathogen attack, hormone signaling, abiotic stresses, or during cell wall organization and plant morphogenesis. Monitoring ROS in plant cells has been limited to biochemical assays and use of fluorescent probes, however, the irreversible oxidation of the fluorescent dyes make it impossible to visualize dynamic changes of ROS. Hyper is a recently developed live cell probe for H2O2 and consists of a circularly permutated YFP (cpYFP) inserted into the regulatory domain of the Escherichia coli hydrogen peroxide (H2O2) sensor OxyR rendering it a H2O2 specific ratiometric, and therefore quantitative probe. Herein, we describe a protocol for using Hyper as a dynamic probe for H2O2 in Arabidopsis with virtually unlimited potential to detect H2O2 throughout the plant and under a broad range of developmental and environmental conditions.


Subject(s)
Hydrogen Peroxide/metabolism , Arabidopsis/cytology , Arabidopsis/genetics , Arabidopsis/metabolism , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Biosensing Techniques , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/genetics , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , NADPH Oxidases/metabolism , Plant Proteins/metabolism , Plant Roots/cytology , Plant Roots/genetics , Plant Roots/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Pollen Tube/cytology , Pollen Tube/genetics , Pollen Tube/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Nicotiana/cytology , Nicotiana/genetics , Nicotiana/metabolism , Transformation, Genetic
7.
Planta ; 234(6): 1163-77, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21744091

ABSTRACT

An indeterminate developmental program allows plant organs to grow continuously by maintaining functional meristems over time. The molecular mechanisms involved in the maintenance of the root apical meristem are not completely understood. We have identified a new Arabidopsis thaliana mutant named moots koom 1 (mko1) that showed complete root apical meristem exhaustion of the primary root by 9 days post-germination. MKO1 is essential for maintenance of root cell proliferation. In the mutant, cell division is uncoupled from cell growth in the region corresponding to the root apical meristem. We established the sequence of cellular events that lead to meristem exhaustion in this mutant. Interestingly, the SCR and WOX5 promoters were active in the mko1 quiescent center at all developmental stages. However, during meristem exhaustion, the mutant root tip showed defects in starch accumulation in the columella and changes in auxin response pattern. Therefore, contrary to many described mutants, the determinate growth in mko1 seedlings does not appear to be a consequence of incorrect establishment or affected maintenance of the quiescent center but rather of cell proliferation defects both in stem cell niche and in the rest of the apical meristem. Our results support a model whereby the MKO1 gene plays an important role in the maintenance of the root apical meristem proliferative capacity and indeterminate root growth, which apparently acts independently of the SCR/SHR and WOX5 regulatory pathways.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/growth & development , Indoleacetic Acids/pharmacology , Meristem/growth & development , Plant Growth Regulators/pharmacology , Plant Roots/growth & development , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Cell Division , Gene Expression Regulation, Plant/genetics , Germination , Homeodomain Proteins/genetics , Meristem/cytology , Meristem/drug effects , Meristem/genetics , Mutation , Phenotype , Plant Roots/cytology , Plant Roots/drug effects , Plant Roots/genetics , Promoter Regions, Genetic/genetics , Seedlings/drug effects , Seedlings/genetics , Seedlings/growth & development , Signal Transduction/genetics , Stem Cell Niche , Transcription Factors/genetics , Transcription Factors/metabolism
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