The proteasome is responsible for caspase-3-like activity during xylem development

Xylem development is a process of xylem cell terminal differentiation that includes initial cell division, cell expansion, secondary cell wall formation and programmed cell death (PCD). PCD in plants and apoptosis in animals share many common characteristics. Caspase-3, which displays Asp-Glu-Val-Asp (DEVD) specificity, is a crucial executioner during animal cells apoptosis. Although a gene orthologous to caspase-3 is absent in plants, caspase-3-like activity is involved in many cases of PCD and developmental processes. However, there is no direct evidence that caspase-3-like activity exists in xylem cell death. In this study, we showed that caspase-3-like activity is present and is associated with secondary xylem development in Populus tomentosa. The protease responsible for the caspase-3-like activity was purified from poplar secondary xylem using hydrophobic interaction chromatography (HIC), Q anion exchange chromatography and gel filtration chromatography. After identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS), it was revealed that the 20S proteasome (20SP) was responsible for the caspase-3-like activity in secondary xylem development. In poplar 20SP, there are seven α subunits encoded by 12 genes and seven β subunits encoded by 12 genes. Pharmacological assays showed that Ac-DEVD-CHO, a caspase-3 inhibitor, suppressed xylem differentiation in the veins of Arabidopsis cotyledons. Furthermore, clasto-lactacystin β-lactone, a proteasome inhibitor, inhibited PCD of tracheary element in a VND6-induced Arabidopsis xylogenic culture. In conclusion, the 20S proteasome is responsible for caspase-3-like activity and is involved in xylem development.     

木质部细胞分化和脱分化的机理

木质部细胞的分化过程包括了密切不可分的细胞程序死亡和次生壁构建两个过程。现在的研究主要是将两个过程分开来研究,各自在细胞生物学和分子生物学上取得了不少进展,有关次生细胞壁方面的研究时间长,成果也较大。有关木质部细胞脱分化的研究相对较少,但也已取得了可喜的进展。     

Programmed cell death in plants: distinguishing between different modes

Programmed cell death (PCD) in plants is a crucial componentof development and defence mechanisms. In animals, differenttypes of cell death (apoptosis, autophagy, and necrosis) havebeen distinguished morphologically and discussed in these morphologicalterms. PCD is largely used to describe the processes of apoptosisand autophagy (although some use PCD and apoptosis interchangeably)while necrosis is generally described as a chaotic and uncontrolledmode of death. In plants, the term PCD is widely used to describemost instances of death observed. At present, there is a vastarray of plant cell culture models and developmental systemsbeing studied by different research groups and it is clear fromwhat is described in this mass of literature that, as with animals,there does not appear to be just one type of PCD in plants.It is fundamentally important to be able to distinguish betweendifferent types of cell death for several reasons. For example,it is clear that, in cell culture systems, the window of timein which ‘PCD’ is studied by different groups varieshugely and this can have profound effects on the interpretationof data and complicates attempts to compare different researcher'sdata. In addition, different types of PCD will probably havedifferent regulators and modes of death. For this reason, inplant cell cultures an apoptotic-like PCD (AL-PCD) has beenidentified that is fairly rapid and results in a distinct corpsemorphology which is visible 4–6 h after release of cytochromec and other apoptogenic proteins. This type of morphology, distinctfrom autophagy and from necrosis, has also been observed inexamples of plant development. In this review, our model systemand how it is used to distinguish specifically between AL-PCDand necrosis will be discussed. The different types of PCD observedin plants will also be discussed and the importance of distinguishingbetween different forms of cell death will be highlighted. Key words: Apoptosis, apoptosis-like programmed cell death (AL-PCD), Arabidopsis, autophagy, mitochondria, necrosis, programmed cell death (PCD) Received 5 June 2007; Revised 13 September 2007 Accepted 20 September 2007     

Precise temporal regulation of roughest is required for correct salivary gland autophagic cell death in Drosophila

The Drosophila roughest (rst) locus encodes an immunoglobulin superfamily transmembrane glycoprotein implicated in a variety of embryonic and postembryonic developmental processes. Here we demonstrate a previously unnoticed role for this gene in the autophagic elimination of larval salivary glands during early pupal stages by showing that overexpression of the Rst protein ectodomain in early pupa leads to persistence of salivary glands up to at least 12 hours after head eversion, although with variable penetrance. The same phenotype is observed in individuals carrying the dominant regulatory allele rst^D, but not in loss of function alleles. Analysis of persistent glands at the ultrastructural level showed that programmed cell death starts at the right time but is arrested at an early stage of the process. Finally we describe the expression pattern and intracellular distribution of Rst in wild type and rst^D mutants, showing that its downregulation in salivary glands at the beginning of pupal stage is an important factor in the correct implementation of the autophagic program of this tissue in space and time. genesis 47:492–504, 2009. © 2009 Wiley‐Liss, Inc.     

Death by over-eating: The Gaucher disease associated gene GBA1, identified in a screen for mediators of autophagic cell death,is necessary for developmental cell death in Drosophila midgut

Autophagy is critical for homeostasis and cell survival during stress, but can also lead to cell death, a little understood process that has been shown to contribute to developmental cell death in lower model organisms, and to human cancer cell death. We recently reported¹ Dasari SK, Bialik S, Levin-Zaidman S, Levin-Salomon V, Merrill AH, Jr., Futerman AH, Kimchi A. Signalome-wide rnai screen identifies gba1 as a positive mediator of autophagic cell death. Cell Death Differ. 2017;24(7):1288-1302. https://doi.org/10.1038/cdd.2017.80. PMID:28574511[Crossref], [PubMed], [Web of Science ®] [Google Scholar] on our thorough molecular and morphologic characterization of an autophagic cell death system involving resveratrol treatment of lung carcinoma cells. To gain mechanistic insight into this death program, we performed a signalome-wide RNAi screen for genes whose functions are necessary for resveratrol-induced death. The screen identified GBA1, the gene encoding the lysosomal enzyme glucocerebrosidase, as an important mediator of autophagic cell death. Here we further show the physiological relevance of GBA1 to developmental cell death in midgut regression during Drosophila metamorphosis. We observed a delay in midgut cell death in two independent Gba1a RNAi lines, indicating the critical importance of Gba1a for midgut development. Interestingly, loss-of-function GBA1 mutations lead to Gaucher Disease and are a significant risk factor for Parkinson Disease, which have been associated with defective autophagy. Thus GBA1 is a conserved element critical for maintaining proper levels of autophagy, with high levels leading to autophagic cell death.     

自噬在细胞存活和死亡中的作用

自噬是亚细胞膜结构发生动态变化并经溶酶体介导对细胞内蛋白质和细胞器降解的过程.通过平衡细胞合成和分解代谢,自噬稳定细胞内环境,维持细胞的存活.然而,过度自噬可导致细胞发生Ⅱ型程序性细胞死亡.自噬与凋亡在细胞死亡过程中的关系十分密切.本文对自噬的过程及其在细胞存活和死亡中的作用作一综述.     

Programmed cell death of follicular epithelium during the late developmental stages of oogenesis in the fruit flies Bactrocera oleae and Ceratitis capitata (Diptera, Tephritidae) is mediated by autophagy

In the present study, we describe the features of programmed cell death of ovarian follicle cells, occurring during the late developmental stages of oogenesis in the olive fruit fly, Bactrocera oleae and the medfly, Ceratitis capitata. During stage 14, the follicle cells contain autophagic vacuoles, and they do not exhibit caspase activity in all parts of the egg chamber. Their nuclei are characterized by condensed chromatin, accompanied with high- but not low-molecular weight DNA fragmentation events exclusively detected in distinct cells of the anterior pole. These data argue for the presence of an autophagy-mediated cell death program in the ovarian follicle cell layer in both species. The above results are likely associated with the abundant phagocytosis observed at the entry of the lateral oviducts, where numerous cell bodies are massively engulfed by epithelial cells. We strongly believe that during the termination of the above Dipteran oogenesis, an efficient mechanism of absorption of the degenerated follicle cells is selectively activated, in order to prevent the blockage of the ovarioles and thus robustly support the physiological completion of the ovulation process.     

Floret-specific differences in gene expression and support for the hypothesis that tapetal degeneration of Zea mays L. Occurs via programmed cell death

The maize (Zea mays) spikelet consists of two florets, each of which contains three developmentally synchronized anthers. Morphologically, the anthers in the upper and lower florets proceed through apparently similar developmental programs. To test for global differences in gene expression and to identify genes that are coordinately regulated during maize anther development, RNA samples isolated from upper and lower floret anthers at six developmental stages were hybridized to eDNA rnicroarrays. Approximately 9% of the tested genes exhibited statistically significant differences in expression between anthers in the upper and lower florets. This finding indicates that several basic biological processes are differentially regulated between upper and lower floret anthers, including metabolism, protein synthesis and signal transduction. Genes that are coordinately regulated across anther development were identified v/a cluster analysis.Analysis of these results identified stage-specific, early in development, late in development and bi-phasic expression profiles. Quantitative RT-PCR analysis revealed that four genes whose homologs in other plant species are involved in programmed cell death are up-regulated just prior to the time the tapetum begins to visibly degenerate (i.e., the mid-microspore stage). This finding supports the hypothesis that developmentally normal tapetal degeneration occurs via programmed cell death.     

New insights into pioneer root xylem development: evidence obtained from Populus trichocarpa plants grown under field conditions

Background and Aims

Effective programmed xylogenesis is critical to the structural framework of the plant root system and its central role in the acquisition and long-distance transport of water and nutrients. The process of xylem differentiation in pioneer roots under field conditions is poorly understood. In this study it is hypothesized that xylogenesis, an example of developmental programmed cell death (PCD), in the roots of woody plants demonstrates a clearly defined sequence of events resulting in cell death. A comprehensive analysis was therefore undertaken to identify the stages of xylogenesis in pioneer roots from procambial cells to fully functional vessels with lignified cell walls and secondary cell wall thickenings.

Methods

Xylem differentiation was monitored in the pioneer roots of Populus trichocarpa at the cytological level using rhizotrons under field conditions. Detection and localization of the signalling molecule nitric oxide (NO) and hydrogen peroxide (H₂O₂) was undertaken and a detailed examination of nuclear changes during xylogenesis was conducted. In addition, analyses of the expression of genes involved in secondary cell wall synthesis were performed in situ.

Key Results

The primary event in initially differentiating tracheary elements (TEs) was a burst of NO in thin-walled cells, followed by H₂O₂ synthesis and the appearance of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling)-positive nuclei. The first changes in nuclear structure were observed in the early stages of xylogenesis of pioneer roots, prior to lignification; however, the nucleus was detectable under transmission electron microscopy in differentiating cells until the stage at which vacuole integrity was maintained, indicating that their degradation was slow and prolonged. The subsequent sequence of events involved secondary cell wall formation and autophagy. Potential gene markers from the cinnamyl alcohol dehydrogenase (CAD) gene family that were related to secondary wall synthesis were associated with primary xylogenesis, showing clear expression in cells that undergo differentiation into TEs and in the thin-walled cells adjacent to the xylem pole.

Conclusions

The early events of TE formation during pioneer root development are described, together with the timing of xylogenesis from signalling via NO, through secondary cell wall synthesis and autophagy events that are initiated long before lignification. This is the first work describing experiments conducted in planta on roots under field conditions demonstrating that the process of xylogenesis in vivo might be gradual and complex.     

Relationship between growth arrest and autophagy in midgut programmed cell death in Drosophila

Autophagy has been implicated in both cell survival and programmed cell death (PCD), and this may explain the apparently complex role of this catabolic process in tumourigenesis. Our previous studies have shown that caspases have little influence on Drosophila larval midgut PCD, whereas inhibition of autophagy severely delays midgut removal. To assess upstream signals that regulate autophagy and larval midgut degradation, we have examined the requirement of growth signalling pathways. Inhibition of the class I phosphoinositide-3-kinase (PI3K) pathway prevents midgut growth, whereas ectopic PI3K and Ras signalling results in larger cells with decreased autophagy and delayed midgut degradation. Furthermore, premature induction of autophagy is sufficient to induce early midgut degradation. These data indicate that autophagy and the growth regulatory pathways have an important relationship during midgut PCD. Despite the roles of autophagy in both survival and death, our findings suggest that autophagy induction occurs in response to similar signals in both scenarios.     

DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death

Death-associated protein kinase (DAPk) and DAPk-related protein kinase (DRP)-1 proteins are Ca+2/calmodulin-regulated Ser/Thr death kinases whose precise roles in programmed cell death are still mostly unknown. In this study, we dissected the subcellular events in which these kinases are involved during cell death. Expression of each of these DAPk subfamily members in their activated forms triggered two major cytoplasmic events: membrane blebbing, characteristic of several types of cell death, and extensive autophagy, which is typical of autophagic (type II) programmed cell death. These two different cellular outcomes were totally independent of caspase activity. It was also found that dominant negative mutants of DAPk or DRP-1 reduced membrane blebbing during the p55/tumor necrosis factor receptor 1-induced type I apoptosis but did not prevent nuclear fragmentation. In addition, expression of the dominant negative mutant of DRP-1 or of DAPk antisense mRNA reduced autophagy induced by antiestrogens, amino acid starvation, or administration of interferon-gamma. Thus, both endogenous DAPk and DRP-1 possess rate-limiting functions in these two distinct cytoplasmic events. Finally, immunogold staining showed that DRP-1 is localized inside the autophagic vesicles, suggesting a direct involvement of this kinase in the process of autophagy.     

Programmed cell death during embryogenesis in maize

Programmed cell death (PCD) in plants is considered an integral part of development. Evidence of DNA fragmentation, occurring at specific sites and times during embryo formation in maize (Zea mays L.), was obtained using terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labelling (TUNEL) and by genomic DNA ladder detection. During the crucial period of elaboration of the primary shoot and root axis (14-20 d after pollination), TUNEL-positive nuclei are present in the scutellum, coleoptile, root cap and principally in the suspensor. Additional evidence of a form of programmed cell death occurring in these tissues comes from the detection of a DNA ladder. Upon completion of the differentiation process, all embryonic cells are TUNEL-negative, indicating that possible programmed cell death events during maize embryogenesis are confined to structures or organs that do not contribute to the adult plant body.