蛋白酶体活性缺失对拟南芥根发育影响的显微和超微结构观察

以拟南芥根为材料,运用光学和透射电子显微镜分析了蛋白酶体抑制剂MG132对拟南芥根尖伸长区细胞的显微及超微结构的影响。结果发现:(1)微分干涉显微镜观察结果表明,MG132处理将导致拟南芥根部伸长区细胞的细胞质液泡化,并且抑制剂浓度越高细胞质液泡化越明显。(2)半薄切片结合考马斯亮蓝染色结果表明,MG132诱导的液泡中富含蛋白质。(3)免疫荧光标记结合共聚焦显微镜观察结果表明,液泡中的蛋白质主要为泛素缀合蛋白,暗示泛素化蛋白质的积累诱导细胞质自体吞噬的发生。(4)透射电镜观察结果表明,MG132处理的确诱导了自体吞噬作用的发生以及随后发生的自噬起源的细胞质液泡化。该研究结果为泛素/蛋白酶体途径与自体吞噬依赖的蛋白降解系统之间的联系提供了线索。     

Roles of the ubiquitin/proteasome pathway in pollen tube growth with emphasis on MG132-induced alterations in ultrastructure, cytoskeleton, and cell wall components

The ubiquitin/proteasome pathway represents one of the most important proteolytic systems in eukaryotes and has been proposed as being involved in pollen tube growth, but the mechanism of this involvement is still unclear. Here, we report that proteasome inhibitors MG132 and epoxomicin significantly prevented Picea wilsonii pollen tube development and markedly altered tube morphology in a dose- and time-dependent manner, while hardly similar effects were detected when cysteine-protease inhibitor E-64 was used. Fluorogenic kinetic assays using fluorogenic substrate sLLVY-AMC confirmed MG132-induced inhibition of proteasome activity. The inhibitor-induced accumulation of ubiquitinated proteins (UbPs) was also observed using immunoblotting. Transmission electron microscopy revealed that MG132 induces endoplasmic reticulum (ER)-derived cytoplasmic vacuolization. Immunogold-labeling analysis demonstrated a significant accumulation of UbPs in degraded cytosol and dilated ER in MG132-treated pollen tubes. Fluorescence labeling with fluorescein isothiocyanate-phalloidin and beta-tubulin antibody revealed that MG132 disrupts the organization of F-actin and microtubules and consequently affects cytoplasmic streaming in pollen tubes. However, tip-focused Ca2+ gradient, albeit reduced, seemingly persists after MG132 treatment. Finally, fluorescence labeling with antipectin antibodies and calcofluor indicated that MG132 treatment induces a sharp decline in pectins and cellulose. This result was confirmed by Fourier transform infrared analysis, thus demonstrating for the first time the inhibitor-induced weakening of tube walls. Taken together, these findings suggest that MG132 treatment promotes the accumulation of UbPs in pollen tubes, which induces ER-derived cytoplasmic vacuolization and depolymerization of cytoskeleton and consequently strongly affects the deposition of cell wall components, providing a mechanistic framework for the functions of proteasome in the tip growth of pollen tubes.     

Root growth, developmental changes in the apex, and hydraulic conductivity for Opuntia ficus-indica during drought

Developmental changes in the root apex and accompanying changes in lateral root growth and root hydraulic conductivity were examined for Opuntia ficus-indica (L.) Miller during rapid drying, as occurs for roots near the soil surface, and more gradual drying, as occurs in deeper soil layers. During 7 d of rapid drying (in containers with a 3-cm depth of vermiculite), the rate of root growth decreased sharply and most root apices died; such a determinate pattern of root growth was not due to meristem exhaustion but rather to meristem mortality after 3 d of drying. The length of the meristem, the duration of the cell division cycle, and the length of the elongation zone were unchanged during rapid drying. During 14 d of gradual drying (in containers with a 6-cm depth of vermiculite), root mortality was relatively low; the length of the elongation zone decreased by 70%, the number of meristematic cells decreased 30%, and the duration of the cell cycle increased by 36%. Root hydraulic conductivity ( L _P) decreased to one half during both drying treatments; L _P was restored by 2 d of rewetting owing to the emergence of lateral roots following rapid drying and to renewed apical elongation following gradual drying. Thus, in response to drought, the apical meristems of roots of O. ficus-indica near the surface die, whereas deeper in the substrate cell division and elongation in root apices continue. Water uptake in response to rainfall in the field can be enhanced by lateral root proliferation near the soil surface and additionally by resumption of apical growth for deeper roots.     

The proteasome is involved in the first metaphase-to-anaphase transition of meiosis in rat oocytes

The proteasome engages in protein degradation as a regulatory process in biological transactions. Among other cellular processes, the proteasome participates in degradation of ubiquinated cyclins in mitosis. However, its role in meiosis has not been established. Resumption of meiosis in the oocyte involves the activation of maturation promoting factor (MPF), a complex of p34cdc2 and cyclin B. Inactivation of this factor, occurring between the two meiotic divisions, is associated with degradation of cyclin B. In this study, we examined the possible involvement of the proteasome in regulation of the exit from metaphase I in spontaneously maturing rat oocytes. We found that upon resumption of meiosis, proteasomes translocate to the spindle apparatus. We further demonstrated that specific inhibitors of proteasome catalytic activity, MG132 and lactacystin, blocked polar body extrusion. Chromosome and microtubule fluorescent staining verified that MG132-treated oocytes were arrested at metaphase I. Intervention of proteasomal action with this inhibitor also resulted in accumulation of cyclin B and elevated activity of MPF. These data demonstrate that proteasomal catalytic activity is absolutely essential for the decrease in MPF activity and completion of the first meiotic division. Its translocation to the spindle apparatus may facilitate the timely degradation of cyclin B.     

Analysis of Cell Division and Elongation Underlying the Developmental Acceleration of Root Growth in Arabidopsis thaliana

To investigate the relation between cell division and expansion in the regulation of organ growth rate, we used Arabidopsis thaliana primary roots grown vertically at 20°C with an elongation rate that increased steadily during the first 14 d after germination. We measured spatial profiles of longitudinal velocity and cell length and calculated parameters of cell expansion and division, including rates of local cell production (cells mm⁻¹ h⁻¹) and cell division (cells cell⁻¹ h⁻¹). Data were obtained for the root cortex and also for the two types of epidermal cell, trichoblasts and atrichoblasts. Accelerating root elongation was caused by an increasingly longer growth zone, while maximal strain rates remained unchanged. The enlargement of the growth zone and, hence, the accelerating root elongation rate, were accompanied by a nearly proportionally increased cell production. This increased production was caused by increasingly numerous dividing cells, whereas their rates of division remained approximately constant. Additionally, the spatial profile of cell division rate was essentially constant. The meristem was longer than generally assumed, extending well into the region where cells elongated rapidly. In the two epidermal cell types, meristem length and cell division rate were both very similar to that of cortical cells, and differences in cell length between the two epidermal cell types originated at the apex of the meristem. These results highlight the importance of controlling the number of dividing cells, both to generate tissues with different cell lengths and to regulate the rate of organ enlargement.     

Constitutive autophagy in plant root cells

In previous studies, using a membrane-permeable protease inhibitor, E-64d, we showed that autophagy occurs constitutively in the root cells of barley and Arabidopsis. In the present study, a fusion protein composed of the autophagy-related protein AtAtg8 and green fluorescent protein (GFP) was expressed in Arabidopsis to visualize autophagosomes. We first confirmed the presence of autophagosomes with GFP fluorescence in the root cells of seedlings grown on a nutrient-sufficient medium. The number of autophagosomes changed as the root cells grew and differentiated. In cells near the apical meristem, autophagosomes were scarcely found. However, a small but significant number of autophagosomes existed in the elongation zone. More autophagosomes were found in the differentiation zone where cell growth ceases but the cells start to form root hair. In addition, we confirmed that autophagy is activated under starvation conditions in Arabidopsis root cells. When the root tips were cultured in a sucrose-free medium, the number of autophagosomes increased in the elongation and differentiation zones, and a significant number of autophagosomes appeared in cells near the apical meristem. The results suggest that autophagy in plant root cells is involved not only in nutrient recycling under nutrient-limiting conditions but also in cell growth and root hair formation.     

Quiescent fibroblasts are protected from proteasome inhibition-mediated toxicity

Proteasome inhibition is used as a treatment strategy for multiple types of cancers. Although proteasome inhibition can induce apoptotic cell death in actively proliferating cells, it is less effective in quiescent cells. In this study, we used primary human fibroblasts as a model system to explore the link between the proliferative state of a cell and proteasome inhibition-mediated cell death. We found that proliferating and quiescent fibroblasts have strikingly different responses to MG132, a proteasome inhibitor; proliferating cells rapidly apoptosed, whereas quiescent cells maintained viability. Moreover, MG132 treatment of proliferating fibroblasts led to increased superoxide anion levels, juxtanuclear accumulation of ubiquitin- and p62/SQSTM1-positive protein aggregates, and apoptotic cell death, whereas MG132-treated quiescent cells displayed fewer juxtanuclear protein aggregates, less apoptosis, and higher levels of mitochondrial superoxide dismutase. In both cell states, reducing reactive oxygen species with N-acetylcysteine lessened protein aggregation and decreased apoptosis, suggesting that protein aggregation promotes apoptosis. In contrast, increasing cellular superoxide levels with 2-methoxyestradiol treatment or inhibition of autophagy/lysosomal pathways with bafilomycin A1 sensitized serum-starved quiescent cells to MG132-induced apoptosis. Thus, antioxidant defenses and the autophagy/lysosomal pathway protect serum-starved quiescent fibroblasts from proteasome inhibition-induced cytotoxicity.     

A role for redox factors in shaping root architecture under phosphorus deficiency

The developmental response of the Arabidopsis root system to low phosphorus (P) availability involves the reduction in primary root elongation accompanied by the formation of numerous lateral roots. We studied the roles of selected redox metabolites, namely, radical oxygen species (ROS) and ascorbic acid (ASC) in the regulation of root system architecture by different P availability. Rapidly growing roots of plants grown on P-sufficient medium synthesize ROS in root elongation zone and quiescent centre. We have demonstrated that the arrest of root elongation at low P medium coincides with the disappearance of ROS from the elongation zone. P-starvation resulted in a decrease in ascorbic acid level in roots. This correlated with a decrease in cell division activity. On the other hand, feeding P-deficient plants with ASC, stimulated mitotic activity in the primary root meristem and partly reversed the inhibition of root growth imposed by low P conditions. In this paper, we discuss the idea of the involvement of redox agents in the regulation of root system architecture under low P availability.Key words: ascorbic acid, phosphate deficiency, primary root, radical oxygen species, root growth, root system architecture     

Proteasome inhibition induces <Emphasis Type="Italic">hsp30</Emphasis> and <Emphasis Type="Italic">hsp70</Emphasis> gene expression as well as the acquisition of thermotolerance in <Emphasis Type="Italic">Xenopus laevis</Emphasis> A6 cells

Previous studies have shown that inhibiting the activity of the proteasome leads to the accumulation of damaged or unfolded proteins within the cell. In this study, we report that proteasome inhibitors, lactacystin and carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132), induced the accumulation of ubiquitinated proteins as well as a dose- and time-dependent increase in the relative levels of heat shock protein (HSP)30 and HSP70 and their respective mRNAs in Xenopus laevis A6 kidney epithelial cells. In A6 cells recovering from MG132 exposure, HSP30 and HSP70 levels were still elevated after 24 h but decreased substantially after 48 h. The activation of heat shock factor 1 (HSF1) may be involved in MG132-induced hsp gene expression in A6 cells since KNK437, a HSF1 inhibitor, repressed the accumulation of HSP30 and HSP70. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. Immunocytochemical studies determined that HSP30 was localized primarily in the cytoplasm of lactacystin- or MG132-treated cells. In some cells treated with higher concentrations of MG132 or lactacystin, we observed in the cortical cytoplasm (1) relatively large HSP30 staining structures, (2) colocalization of actin and HSP30, and (3) cytoplasmic areas that were devoid of HSP30. Lastly, MG132 treatment of A6 cells conferred a state of thermotolerance such that they were able to survive a subsequent thermal challenge.     

Restoration of cell growth and proliferation in the wheat roots after their inhibition by nickel sulfate

The dynamics of cell growth and proliferation restoration in different tissues and quiescent center (QC) in the wheat (Triticum aestivum L.) seedling roots and also the differentiation of rhizodermal cells and lateral root initiation after 48-h treatment with 100 μM NiSO₄ were studied. Within 24 h after nickel removal from medium, root growth was resumed due to the increase in the rate of cell growth in the meristem and the region where cell elongation started in control roots. Stimulation of cell proliferation was restored in the main part of the meristem and later in the initial cells of the files and QC. Cell proliferation was not observed in the QC. The time of cell proliferation resumption in the roots and in tested tissues depended on the degree of their injury by nickel treatment. In most tested roots, DNA synthesis and cell division were restored in 32 h. In the cells leaving the meristem due to the resumption of their growth and proliferation, growth of root hairs started. In 48 h, the number of roots with perished cells in the rhizodermis in the meristem was sharply increased and the regeneration of the damaged region by the cells of outer cortex was observed. Only after the appearance of root hairs, the cells coming from the meristem started to elongate. In most roots, the formation of the new elongation zone occurred in 56 h. During its formation, the initiation of lateral root primordia was shifted in the basipetal direction. It was concluded that the cessation of cell growth and proliferation under the influence of high concentration of heavy metal (HM) ions is not lethal for the root. At the action of toxic HM concentrations, the plant strategy is the maintenance of meristematic cell capacity for cell growth and proliferation resumption. The cellular mechanism of this capacity maintenance is the transition of meristematic cells from G₁ phase to dormancy due to growth inhibition and the inhibition of the transition to DNA synthesis.     

Stunted plant 1 mediates effects of cytokinin, but not of auxin, on cell division and expansion in the root of Arabidopsis

Plants control organ growth rate by adjusting the rate and duration of cell division and expansion. Surprisingly, there have been few studies where both parameters have been measured in the same material, and thus we have little understanding of how division and expansion are regulated interdependently. We have investigated this regulation in the root meristem of the stunted plant 1 (stp1) mutation of Arabidopsis, the roots of which elongate more slowly than those of the wild type and fail to accelerate. We used a kinematic method to quantify the spatial distribution of the rate and extent of cell division and expansion, and we compared stp1 with wild type and with wild type treated with exogenous cytokinin (1 microM zeatin) or auxin (30 nM 2,4-dichlorophenoxyacetic acid). All treatments reduced average cell division rates, which reduced cell production by the meristem. Auxin lowered root elongation by narrowing the elongation zone and reducing the time spent by a cell in this zone, but did not decrease maximal strain rate. In addition, auxin increased the length of the meristem. In contrast, cytokinin reduced root elongation by lowering maximal strain rate, but did not change the time spent by a cell within the elongation zone; also, cytokinin blocked the increase in length and cell number of the meristem and elongation zone. The cytokinin-treated wild type phenocopied stp1 in nearly every detail, supporting the hypothesis that cytokinin affects root growth via STP1. The opposite effects of auxin and cytokinin suggest that the balance of these hormones may control the size of the meristem.     

Temperature‐compensated cell production rate and elongation zone length in the root of Arabidopsis thaliana

To understand how root growth responds to temperature, we used kinematic analysis to quantify division and expansion parameters in the root of Arabidopsis thaliana. Plants were grown at temperatures from 15 to 30 °C, given continuously from germination. Over these temperatures, root length varies more than threefold in the wild type but by only twofold in a double mutant for phytochrome‐interacting factor 4 and 5. For kinematics, the spatial profile of velocity was obtained with new software, Stripflow. We find that 30 °C truncates the elongation zone and curtails cell production, responses that probably reflect the elicitation of a common pathway for handling severe stresses. Curiously, rates of cell division at all temperatures are closely correlated with rates of radial expansion. Between 15 to 25 °C, root growth rate, maximal elemental elongation rate, and final cell length scale positively with temperature whereas the length of the meristem scales negatively. Non‐linear temperature scaling characterizes meristem cell number, time to transit through either meristem or elongation zone, and average cell division rate. Surprisingly, the length of the elongation zone and the total rate of cell production are temperature invariant, constancies that have implications for our understanding of how the underlying cellular processes are integrated.     

Induction of autophagy by proteasome inhibitor is associated with proliferative arrest in colon cancer cells

The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Blockade of UPS by proteasome inhibitors has been shown to activate autophagy. Recent evidence also suggests that proteasome inhibitors may inhibit cancer growth. In this study, the effect of a proteasome inhibitor MG-132 on the proliferation and autophagy of cultured colon cancer cells (HT-29) was elucidated. Results showed that MG-132 inhibited HT-29 cell proliferation and induced G₂/M cell cycle arrest which was associated with the formation of LC3⁺ autophagic vacuoles and the accumulation of acidic vesicular organelles. MG-132 also increased the protein expression of LC3-I and -II in a time-dependent manner. In this connection, 3-methyladenine, a Class III phosphoinositide 3-kinase inhibitor, significantly abolished the formation of LC3⁺ autophagic vacuoles and the expression of LC3-II but not LC3-I induced by MG-132. Taken together, this study demonstrates that inhibition of proteasome in colon cancer cells lowers cell proliferation and activates autophagy. This discovery may shed a new light on the novel function of proteasome in the regulation of autophagy and proliferation in colon cancer cells.     

Functional characterization of NtCDPK1 in tobacco

We previously showed that NtCDPK1, a tobacco cal-cium-dependent protein kinase, interacts with and phosphorylates the Rpn3 regulatory subunit of the 26S proteasome, and that both NtCDPK1 and Rpn3 are mainly expressed in rapidly proliferating tissues, in-cluding shoot and root meristem. In this study, we ex-amined NtCDPK1 expression in roots using GUS ex-pression in transgenic Arabidopsis plants, and investi-gated its function in root development by generating transgenic tobacco plants carrying a sense NtCDPK1 transgene. GUS activity was first detected in roots two days after sowing. In later stages, strong GUS expres-sion was detected in the root meristem and elongation zone, as well as the initiation sites and branch points of lateral roots. Transgenic tobacco plants in which NtCDPK1 expression was suppressed were smaller, and their root development was abnormal, with reduced lateral root formation and less elongation. These re-sults suggest that NtCDPK1 plays a role in a signaling pathway regulating root development in tobacco.     

Caspase-8 dependent osteosarcoma cell apoptosis induced by proteasome inhibitor MG132

Many researchers have reported that proteasome inhibitors could induce apoptosis in a variety of cancer cells, such as breast cancer cell, lung cancer cell, and lymphoma cell. However, the effect of proteasome inhibitors on osteocsarcoma cells and the mechanisms are seldom studied. In this study, we found proteasome inhibitor MG132 was an effective inducer of apoptosis in human osteosarcoma MG-63 cells. On normal human diploid fibroblast cells, MG132 did not show any apoptosis-inducing effects. Apoptotic changes such as DNA fragment and apoptotic body were observed in MG132-treated cells and MG132 mostly caused MG-63 cell arrest at G(2)-M-phase by cell cycle analysis. Increased activation of caspase-8, accumulation of p27(Kip1), and an increased ratio of Bax:Bcl-2 were detected by RT-PCR and Western blot analysis. Activation of caspase-3 and caspase-9 were not observed. This suggests that the apoptosis induced by MG132 in MG63 cells is caspase-8 dependent, p27 and bcl-2 family related.     

Regions of differential cell elongation and mitosis, and root meristem morphology in different tissues of geotropically stimulated maize root apices

We examined cell length, mitosis, and root meristem “cuticle” in different tissues of geostimulated, red light-exposed primary roots of corn (Zea Mays, Wisconsin hybrid 64A × 22R). The examination was done at 15-minute intervals for a period of 240 minutes. Differences in cell elongation between the upper and lower sides were most prominent between 1.5 and 2.5 mm from the root meristem; the outer cortex had the greatest elongation growth, and the upper cells showed a significant increase in length compared to the lower. A differential mitosis was also found, with the lower tissue being greater. We infer that the mitotic activity is indicative of cell division, and this division occurs strictly in the first 1.5 mm of the root meristem. The combined effect of differential cell elongation and cell division results in the localization of the geotropic curvature in the 1.5- to 2.5-mm region from the root meristem. Mitosis that occurs primarily in the cortex and stele were asynchronous; the peak of cortical division preceded that of the stele. Both peaks occurred before the peak of geotropism. A densely stained layer separates the cap from the root meristem. This layer is thinner at the apex of the root meristem. The area of the thin region increased with time and peaked at 180 minutes after geostimulation, which was coincidental with the peak of the geotropic response.     

多胺生物合成抑制剂D-精氨酸对拟南芥幼苗根系生长的影晌

为探讨多胺生物合成抑制剂D-精氨酸（D-arginine,D-Arg）对拟南芥根系生长的影响,首先用腐胺（0．1mmol‘L-1）和D—Arg（1．0mmol·L-1）处理种子萌发后生长2d的拟南芥幼苗。腐胺（0．1mmol·L-1）显著促进主根伸长,D-Arg（1．0mmol-L-1）显著抑制主根伸长,并对主根根尖的细胞形态有明显影响。为了进一步了解D—Arg影响拟南芥主根生长的机理,采用浓度梯度D．Arg处理幼苗根系。实验结果表明,随着D-Arg浓度增加（0．2～1．0mmol·L-1）,拟南芥幼苗主根生长受抑制的程度越严重。微分干涉观察主根根尖发现,外源施加D—Arg,引起拟南芥主根根尖分生区的细胞数目减少,使拟南芥幼苗表现出主根的伸长生长变缓。当分生区数目较少时,出现主根几乎不再仲长的现象。由此推测,多胺生物合成抑制剂D-Arg对拟南芥幼苗根生长的抑制作用机制,是D-Arg影响了其根尖分生区的细胞分裂活动,使分生区细胞数目减少,从而引起分生区长度减小,最终导致拟南芥主根仲长生长受到抑制。     

Interaction of Epiphytic Bacteria and Activated Carbon on Root Growth

The influence of activated carbon and aseptic conditions has been studied on the growth of the primary root of wheat seedlings in order to ascertain whether or not the growth effect of activated carbon is connected with the occurrence of epiphytic bacteria. Growth was measured as mitotic activity, rate of cell elongation and duration of cell elongation. The surface infection of the septic roots probably consisted of common airborn and waterborn bacteria. Aseptic conditions increased the rate of cell elongation by ca 70 % but had no effect on the meristem activity. Activated carbon increased mitoses in the meristem and slightly augmented the duration of cell elongation but had no effect on the rate of elongation. The effects of sepsis and carbon were independent and appeared additative. Activated carbon removed inhibitors produced by the root tip itself but not those formed by the bacteria. In these experiments neither group of inhibitors seemed to contain IAA-like substances.