Reverse genetic and cell biological approaches to the study of developmental functions of Class XI myosin in Arabidopsis thaliana

Myosin proteins function as molecular motors that drive the ATP-dependent movement of cellular components along actin filaments. Vascular plants encode two different types of myosin, referred to as class VIII and class XI. Although class XI myosins have been suggested to function in organelle movement and cytoplasmic streaming, little is known about their cellular function in detail. The Arabidopsis genome encodes 13 class XI myosin genes. The reasons for the relatively large number of myosin XI isoforms present within a single plant species are unknown. To investigate the function of these gene products in the cell, we determined the spatial and temporal gene expression patterns by constructing promoter-reporter plants. Myosin genes are expressed in a variety of tissues with substantial overlap between family members. To study the biological function more intensively, homozygous T-DNA insertion lines were isolated for all 13 genes. Interestingly, five mutants showed phenotypes related to root hairs. mya2, xi-b, and xi-k showed shorter root hairs than in wild type while xi-h and mya1 produced a higher density of root hairs on the epidermis. MYA1 and XI-K are the most similar isoforms among the 13 myosins and their double mutant showed an additive phenotype with extremely short root hairs suggesting that these two myosins have partially redundant functions. Interestingly organelle movements, especially those of peroxisomes, were reduced in mya1 xi-k. Tip growth is the key growth mechanism in root hairs and pollen tubes. Many kinds of vesicles are trafficking toward (or backward from) the apical dome of root hairs to supply membrane and cell wall material as well as energy for growing tips. These movements along the shank of the hair occurred with velocities around 2 to 3 μm/s for Arabidopsis thaliana. In xi-k mutants, root hairs grew more slowly and terminated sooner than in wild type. Interestingly, this reduction of growth rate was correlated with a fluctuation of YFP-RAbA4b accumulation at the tip of growing root hairs. Other markers, including PI4P lipid and ER, as well as calcium and actin dynamics did not show significant differences. A YFP-XI-K construct driven by its native promoter could rescue the mutant phenotype and revealed accumulation of this myosin in the tip of growing root hairs. The distribution of YFP-XI-K in the root hair tip partially overlapped with CFP-RHD4-labeled vesicles at the subapex and YFP-RabA4b vesicles at the apex of root hairs, suggesting that myosin XI-K might be involved in the accumulation of unidentified vesicles in the tip of growing root hairs. Characterization of two mutants that showed ectopic root hair growth in the epidermis, resulting in a higher density of root hairs than wild type, mya1 and xi-h, were initiated with two analyses. At first, staining pattern of promoter-reporter constructs of three key transcription factors, WER, EGL3, and GL2 were observed in mya1. Although variation in individual samples was too large to conclude, GL2 staining patterns in mya1 occasionally were unorganized. Increasing sample population and detail study is necessary. Secondly, effects of phosphate deficiency were observed with the mya1 and the xi-h in series of phosphate concentrations ranging from 1μM to 300μM. The xi-h mutant showed insensitivity on root hair production upon phosphate deficiency, suggesting a potential function of XI-H in the response to phosphate deficiency. Confirmation of these results and further study of the MYA1 and the XI-H is essential. In summary, this study established a systematic approach to investigate the biological function of class XI myosins in plant development and significantly increases our understanding of the function of XI-K myosin in root hair tip growth

Myosin XIK of Arabidopsis thaliana Accumulates at the Root Hair Tip and Is Required for Fast Root Hair Growth

Myosin motor proteins are thought to carry out important functions in the establishment and maintenance of cell polarity by moving cellular components such as organelles, vesicles, or protein complexes along the actin cytoskeleton. In Arabidopsis thaliana, disruption of the myosin XIK gene leads to reduced elongation of the highly polar root hairs, suggesting that the encoded motor protein is involved in this cell growth. Detailed live-cell observations in this study revealed that xik root hairs elongated more slowly and stopped growth sooner than those in wild type. Overall cellular organization including the actin cytoskeleton appeared normal, but actin filament dynamics were reduced in the mutant. Accumulation of RabA4b-containing vesicles, on the other hand, was not significantly different from wild type. A functional YFP-XIK fusion protein that could complement the mutant phenotype accumulated at the tip of growing root hairs in an actin-dependent manner. The distribution of YFP-XIK at the tip, however, did not match that of the ER or several tip-enriched markers including CFP-RabA4b. We conclude that the myosin XIK is required for normal actin dynamics and plays a role in the subapical region of growing root hairs to facilitate optimal growth. DOI: 10.1371/journal.pone.007674

The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species

Background Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes. Results We present a novel transient assay based on cocultivation of young Arabidopsis (Arabidopsis thaliana) seedlings with Agrobacterium tumefaciens in the presence of a surfactant which does not require any dedicated equipment and can be carried out within one week from sowing seeds to protein analysis. This Fast Agro-mediated Seedling Transformation (FAST) was used successfully to express a wide variety of constructs driven by different promoters in Arabidopsis seedling cotyledons (but not roots) in diverse genetic backgrounds. Localizations of three previously uncharacterized proteins were identified by cotransformation with fluorescent organelle markers. The FAST procedure requires minimal handling of seedlings and was also adaptable for use in 96-well plates. The high transformation efficiency of the FAST procedure enabled protein detection from eight transformed seedlings by immunoblotting. Protein-protein interaction, in this case HY5 homodimerization, was readily detected in FAST-treated seedlings with Förster resonance energy transfer and bimolecular fluorescence complementation techniques. Initial tests demonstrated that the FAST procedure can also be applied to other dicot and monocot species, including tobacco, tomato, rice and switchgrass. Conclusion The FAST system provides a rapid, efficient and economical assay of gene function in intact plants with minimal manual handling and without dedicated device. This method is potentially ideal for future automated high-throughput analysis

The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species

Background Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes. Results We present a novel transient assay based on cocultivation of young Arabidopsis (Arabidopsis thaliana) seedlings with Agrobacterium tumefaciens in the presence of a surfactant which does not require any dedicated equipment and can be carried out within one week from sowing seeds to protein analysis. This Fast Agro-mediated Seedling Transformation (FAST) was used successfully to express a wide variety of constructs driven by different promoters in Arabidopsis seedling cotyledons (but not roots) in diverse genetic backgrounds. Localizations of three previously uncharacterized proteins were identified by cotransformation with fluorescent organelle markers. The FAST procedure requires minimal handling of seedlings and was also adaptable for use in 96-well plates. The high transformation efficiency of the FAST procedure enabled protein detection from eight transformed seedlings by immunoblotting. Protein-protein interaction, in this case HY5 homodimerization, was readily detected in FAST-treated seedlings with Förster resonance energy transfer and bimolecular fluorescence complementation techniques. Initial tests demonstrated that the FAST procedure can also be applied to other dicot and monocot species, including tobacco, tomato, rice and switchgrass. Conclusion The FAST system provides a rapid, efficient and economical assay of gene function in intact plants with minimal manual handling and without dedicated device. This method is potentially ideal for future automated high-throughput analysis

Stromule extension along microtubules coordinated with actin-mediated anchoring guides perinuclear chloroplast movement during innate immunity.

Dynamic tubular extensions from chloroplasts called stromules have recently been shown to connect with nuclei and function during innate immunity. We demonstrate that stromules extend along microtubules (MTs) and MT organization directly affects stromule dynamics since stabilization of MTs chemically or genetically increases stromule numbers and length. Although actin filaments (AFs) are not required for stromule extension, they provide anchor points for stromules. Interestingly, there is a strong correlation between the direction of stromules from chloroplasts and the direction of chloroplast movement. Stromule-directed chloroplast movement was observed in steady-state conditions without immune induction, suggesting it is a general function of stromules in epidermal cells. Our results show that MTs and AFs may facilitate perinuclear clustering of chloroplasts during an innate immune response. We propose a model in which stromules extend along MTs and connect to AF anchor points surrounding nuclei, facilitating stromule-directed movement of chloroplasts to nuclei during innate immunity

Spatial chloroplast-to-nucleus signalling involving plastid-nuclear complexes and stromules

Communication between chloroplasts and the nucleus in response to various environmental cues may be mediated by various small molecules. Signalling specificity could be enhanced if the physical contact between these organelles facilitates direct transfer and prevents interference from other subcellular sources of the same molecules. Plant cells have plastidnuclear complexes, which provide close physical contact between these organelles. plastidnuclear complexes have been proposed to facilitate transfer of photosynthesis-derived H₂O₂ to the nucleus in high light. Stromules (stroma filled tubular plastid extensions) may provide an additional conduit for transfer of a wider range of signalling molecules, including proteins. However, plastid-nuclear complexes and stromules have been hitherto treated as distinct phenomena. We suggest that plastid-nuclear complexes and stromules work in a coordinated manner so that, according to environmental conditions or developmental state the two modes of connection contribute to varying extents. We hypothesise that this association is dynamic and that there may be a link between plastid-nuclear complexes and the development of stromules. Furthermore, the changes in contact could alter signalling specificity by allowing an extended or different range of signalling molecules to be delivered to the nucleus

Inhibitory Effect of Yongdamsagan-Tang Water Extract, a Traditional Herbal Formula, on Testosterone-Induced Benign Prostatic Hyperplasia in Rats

Yongdamsagan-tang, a traditional herbal formula, is used widely for the treatment of inflammation and viral diseases. In this study, we investigated whether Yongdamsagan-tang water extract (YSTE) affects testosterone propionate- (TP-) induced benign prostatic hyperplasia (BPH) in a rat model. To induce BPH, rats were injected subcutaneously with 10 mg/kg of TP every day. YSTE was administrated daily by oral gavage at doses of 200 and 500 mg/kg along with the TP injection. After 4 weeks, prostates were collected, weighed, and analyzed. The relative prostrate weight was significantly lower in both YSTE groups (200 and 500 mg/kg/day) compared with the TP-induced BPH group. YSTE administration reduced the expression of proliferation markers PCNA, cyclin D1, and Ki-67 and the histological abnormalities observed in the prostate in TP-induced BPH rats. YSTE attenuated the increase in the TP-induced androgen concentration in the prostate. The YSTE groups also showed decreased lipid peroxidation and increased glutathione reductase activity in the prostate. These findings suggest that YSTE effectively prevented the development of TP-induced BPH in rats through antiproliferative and antioxidative activities and might be useful in the clinical treatment of BPH

Association of pre-operative medication use with post-operative delirium in surgical oncology patients receiving comprehensive geriatric assessment

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.Abstract Background Older patients undergoing surgery tend to have a higher frequency of delirium. Delirium is strongly associated with poor surgical outcomes. This study evaluated the association between pre-operative medication use and post-operative delirium (POD) in surgical oncology patients receiving comprehensive geriatric assessment (CGA). Methods A total of 475 patients who were scheduled for cancer surgery and received CGA from January 2014 to June 2015 were included. Pre-operative medication review through CGA was conducted on polypharmacy (≥5 medications), delirium-inducing medications (DIMs), fall-inducing medications (FIMs), and potentially inappropriate medications (PIMs). POD was confirmed by psychiatric consultation, and DSM-V criteria were used for diagnosing delirium. The model fit of the prediction model was assessed by computing the Hosmer-Lemeshow goodness-of-fit test. Effect size was measured using the Nagelkerke R2. Discrimination of the model was assessed by an analysis of the area under receiver operating curve (AUROC). Results Two models were constructed for multivariate analysis based on univariate analysis; model I included dementia and DIM in addition to age and sex, and model II included PIM instead of DIM of model I. Every one year increase of age increased the risk of POD by about 1.1-fold. DIM was a significant factor for POD after adjusting for confounders (AOR 12.78, 95 % CI 2.83-57.74). PIM was also a significant factor for POD (AOR 5.53, 95 % CI 2.03-15.05). The Hosmer-Lemeshow test results revealed good fits for both models (χ2 = 3.842, p = 0.871 for model I and χ2 = 8.130, p = 0.421 for model II). The Nagelkerke R2 effect size and AUROC for model I was 0.215 and 0.833, respectively. Model II had the Nagelkerke R2effect size of 0.174 and AUROC of 0.819. Conclusions These results suggest that pharmacists comprehensive review for pre-operative medication use is critical for the post-operative outcomes like delirium in older patients