Autophagy in plants

Autophagy is a highly conserved processing mechanism in eukaryotes whereby cytoplasmic components are engulfed in double-membrane vesicles called autophagosomes and are delivered into organelles such as lysosomes (mammal) or vacuoles (yeast/plant) for degradation and recycling of the resulting molecules. Isolation of yeastAUTOPHAGY (ATG) genes has facilitated the identification of correspondingArabidopsis ATG genes based on sequence similarity. Genetic and molecular analyses using knockout and/or knockdown mutants of those genes have unraveled the biological functions of autophagy during plant development, nutrient recycling, and environmental stress responses. Additional roles for autophagy have been suggested in the degradation of oxidized proteins during oxidative stress and the regulation of hypersensitive response (HR)-programmed cell death (PCD) during innate immunity. Our review summarizes knowledge about the structure and function of autophagic pathways andATG components, and the biological roles of autophagy in plants.     

我国科学家在程序性细胞死亡机制研究领域取得重大突破

程序性细胞死亡在调控植物发育和胁迫响应中具有重要作用, 而活性氧是导致程序性细胞死亡的关键因子。日前, 中科院遗传与发育所李家洋研究组对活性氧调控程序性细胞死亡的分子机制进行了深度解析, 首次阐明了苹果酸作为信号分子, 经由叶绿体-线粒体穿梭途径而引发活性氧产生, 继而导致细胞死亡。该研究成果是程序性细胞死亡调控机制领域的重大突破。     

NtProRP1, a novel proline‐rich protein,is an osmotic stress‐responsive factor and specifically functions in pollen tube growth and early embryogenesis in Nicotiana tabacum

Proline‐rich proteins (PRPs) are known to play important roles in sexual plant reproduction. Most of the known proteins in the family were found in styles or pollen and modulate pollen tube growth. Here, we identified a novel member of the gene family, NtProRP1, which is preferentially expressed in tobacco pollen grains, pollen tubes and zygotes. NtProRP1 could be secreted into the extracellular space including the cell wall, and the predicted N‐terminal signal peptide is crucial for its secretion. In NtProRP1‐RNAi plants, pollen germination and pollen tube growth were significantly slower and showed zigzag or swell morphology in vitro. Early embryogenesis also exhibited aberrant development, indicative of its critical role in both pollen tube growth and early embryogenesis. Further investigation revealed that NtProRP1 plays a crucial role in osmotic stress response during pollen tube growth and is likely regulated by Tsi, a stress‐responsive gene, suggesting that the regulatory mechanism is also involved in the stress response during sexual plant reproduction. These data provide evidence that NtProRP1 functions as a downstream factor of Tsi1 in the stress response and converges the stress signal into the modulation of pollen tube growth and early embryogenesis.     

活性氧对植物自噬调控的研究进展

自噬是一种在真核生物中高度保守的降解细胞组分的生物过程, 在饥饿、衰老和病菌感染等过程中起关键作用。而活性氧是有氧生物在正常或胁迫条件下产生的一种代谢副产物, 在植物的生长发育、胁迫适应和程序性细胞死亡过程中起重要作用。最新研究结果表明, 当植物受到病菌感染产生超敏反应时活性氧和自噬在程序性细胞死亡、生长发育和胁迫适应过程中起重要调控作用。因此, 该文结合最新的研究进展, 从活性氧的种类及特点、自噬的分子基础以及活性氧在植物自噬中的作用等方面, 探讨了活性氧与植物自噬之间的信号转导关系。     

Programmed cell death may act as a surveillance mechanism to safeguard male gametophyte development in Arabidopsis

Programmed cell death (PCD) plays an important role in plant growth and development as well as in stress responses. During male gametophyte development, it has been proposed that PCD may act as a cellular surveillance mechanism to ensure successful progression of male gametogenesis, and this suicide protective machinery is repressed under favorable growth conditions. However, the regulatory mechanism of male gametophyte-specific PCD remains unknown. Here, we report the use of a TdT-mediated dUTP nick-end labeling-based strategy for genetic screening of Arabidopsis mutants that present PCD phenotype during male gametophyte development. By using this approach, we identified 12 mutants, designated as pcd in male gametogenesis (pig). pig mutants are defective at various stages of male gametophyte development, among which nine pig mutants show a microspore-specific PCD phenotype occurring mainly around pollen mitosis I or the bicellular stage. The PIG1 gene was identified by map-based cloning, and was found to be identical to ATAXIA TELANGIECTASIA MUTATED (ATM), a highly conserved gene in eukaryotes and a key regulator of the DNA damage response. Our results suggest that PCD may act as a general mechanism to safeguard the entire process of male gametophyte development.     

被子植物有性生殖过程中的细胞程序死亡

细胞程序死亡是植物发育过程中的一种普遍现象。早就认识到高等植物生殖器官中一些细胞的死亡对植物有性生殖具有重要作用。这些细胞的死亡过程与动物组织中的细胞程序死亡基本相同。但植物体内诱导生殖细胞程序死亡的信号及其传导系统则显示出其特点 ,有些还表现出雌、雄性细胞的相互作用。探索植物生殖过程中的细胞程序死亡现象将有利于澄清植物生殖过程中的一些机理问题 ,使过去的细胞学研究结果深入到分子水平进行探讨     

Modulation of intracellular ROS levels by TIGAR controls autophagy

The p53‐inducible TIGAR protein functions as a fructose‐2,6‐bisphosphatase, promoting the pentose phosphate pathway and helping to lower intracellular reactive oxygen species (ROS). ROS functions in the regulation of many cellular responses, including autophagy—a response to stress conditions such as nutrient starvation and metabolic stress. In this study, we show that TIGAR can modulate ROS in response to nutrient starvation or metabolic stress, and functions to inhibit autophagy. The ability of TIGAR to limit autophagy correlates strongly with the suppression of ROS, with no clear effects on the mTOR pathway, and is p53 independent. The induction of autophagy in response to loss of TIGAR can function to moderate apoptotic response by restraining ROS levels. These results reveal a complex interplay in the regulation of ROS, autophagy and apoptosis in response to TIGAR expression, and shows that proteins similar to TIGAR that regulate glycolysis can have a profound effect on the autophagic response through ROS regulation.     

Regulation of mitochondrial NAD pool via NAD+ transporter 2 is essential for matrix NADH homeostasis and ROS production in Arabidopsis

Reactive oxygen species(ROS) play a crucial role in numerous biological processes in plants, including development, responses to environmental stimuli, and programmed cell death(PCD). Deficiency in MOSAIC DEATH 1(MOD1), a plastid-localized enoyl-ACP reductase essential for de novo fatty acid biosynthesis in Arabidopsis thaliana, leads to the increased malate export from chloroplasts to mitochondria, and the subsequent accumulation of mitochondria-generated ROS and PCD. In this study, we report the identification and characterization of a mod1 suppressor, som592. SOM592 encodes mitochondrion-localized NAD~+ transporter 2(NDT2). We show that the mitochondrial NAD pool is elevated in the mod1 mutant. The som592 mutation fully suppressed mitochondrial NADH hyper-accumulation, ROS production, and PCD in the mod1 mutant, indicating a causal relationship between mitochondrial NAD accumulation and ROS/PCD phenotypes. We also show that in wild-type plants, the mitochondrial NAD+uptake is involved in the regulation of ROS production in response to continuous photoperiod. Elevation of the alternative respiration pathway can suppress ROS accumulation and PCD in mod1, but leads to growth restriction. These findings uncover a regulatory mechanism for mitochondrial ROS production via NADH homeostasis in Arabidopsis thaliana that is likely important for growth regulation in response to altered photoperiod.     

Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress

Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H₂DCFDA‐staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into ‘low‐ROS’ and ‘high‐ROS’ subpopulations. Pollen germination assays following fluorescence‐activated cell sorting revealed that the high‐ROS pollen germinated with a frequency that was 35‐fold higher than the low‐ROS pollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose‐dependent alterations in DCF‐fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increased ROS levels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry‐based approaches to investigate metabolic changes during stress responses in pollen.     

Self-consuming innate immunity in Arabidopsis

Programmed cell death (PCD) associated with the pathogen-induced hypersensitive response (HR) is a hallmark of plant innate immunity. HR PCD is triggered upon recognition of pathogen effector molecules by host immune receptors either directly or indirectly via effector modulation of host targets. However, it has been unclear by which molecular mechanisms plants execute PCD during innate immune responses. We recently examined HR PCD in autophagy-deficient Arabidopsis knockout mutants (atg) and find that PCD conditioned by one class of plant innate immune receptors is suppressed in atg mutants. Intriguingly, HR triggered by another class of immune receptors with different genetic requirements is not compromised, indicating that only a specific subset of immune receptors engage the autophagy pathway for HR execution. Thus, our work provides a primary example of autophagic cell death associated with innate immune responses in eukaryotes as well as of pro-death functions for the autophagy pathway in plants.     

Accelerated cell death in Podospora autophagy mutants

Although autophagy is characteristic of type II programmed cell death (PCD), its role in cell death is currently debated. Both cell death-promoting and prosurvival roles of autophagy have been reported depending on the organism and the cell type. In filamentous fungi, a cell death reaction known as an incompatibility reaction occurs when cells of unlike genotype fuse. Cell death by incompatibility is characterized by a dramatic vacuolar enlargement and cell lysis. In Podospora anserina, autophagy is induced early during this cell death reaction. Cell death by incompatibility in Podospora is a model of type II PCD used here to assess the role of autophagy in this type of cell death. We have inactivated PaATG1, the Podospora ortholog of the Saccharomyces cerevisiae ATG1 gene involved in the early steps of autophagy in yeast. The DeltaPaATG1 mutant displays developmental defects characteristic of abrogated autophagy in Podospora. Using the green fluorescent protein-PaATG8 autophagosome marker, we show that autophagy is abolished in this mutant. Neither cell death by incompatibility nor vacuolization are suppressed in DeltaPaATG1 and DeltaPaATG8 autophagy mutants, indicating that a vacuolar cell death reaction without autophagy occurs in Podospora. Our results thus provide a novel example of a type II PCD reaction in which autophagy is not the cause of cell death. In addition, we found that cell death is accelerated in DeltaPaATG null mutants, suggesting that autophagy has a protective role in this type II PCD reaction.     

Classes of programmed cell death in plants, compared to those in animals

Relatively little is known about programmed cell death (PCD) in plants. It is nonetheless suggested here that tonoplast rupture and the subsequent rapid destruction of the cytoplasm can distinguish two large PCD classes. One class, which is here called 'autolytic', shows this feature, whilst the second class (called 'non-autolytic') can include tonoplast rupture but does not show the rapid cytoplasm clearance. Examples of the 'autolytic' PCD class mainly occur during normal plant development and after mild abiotic stress. The 'non-autolytic' PCD class is mainly found during PCD that is due to plant-pathogen interactions. Three categories of PCD are currently recognized in animals: apoptosis, autophagy, and necrosis. An attempt is made to reconcile the recognized plant PCD classes with these groups. Apoptosis is apparently absent in plants. Autophagic PCD in animals is defined as being accompanied by an increase in the number of autophagosomes, autolysosomes, and small lytic vacuoles produced by autolysosomes. When very strictly adhering to this definition, there is no (proof for) autophagic PCD in plants. Upon a slightly more lenient definition, however, the 'autolytic' class of plant PCD can be merged with the autophagic PCD type in animal cells. The 'non-autolytic' class of plant PCD, as defined here, can be merged with necrotic PCD in animals.     

Salt stress-induced programmed cell death in tobacco protoplasts is mediated by reactive oxygen species and mitochondrial permeability transition pore status

The status of mitochondrial permeability transition pore (PTP) and levels of reactive oxygen species (ROS) play key roles in regulating apoptosis in animal cells. To investigate if the PTP and cellular oxidation-reduction state are also involved in salt stress-induced programmed cell death (PCD) in tobacco (Nicotiana tabacum, cultivar BY-2) protoplasts, flow cytometry was used to simultaneously monitor ROS levels, PTP status and PCD. Increased ROS and decreased mitochondrial membrane potential (delta psi(m)) were observed before the appearance of PCD. Pre-treatment with an inhibitor of the PTP opening, cyclosporin A (CsA), effectively retarded the onset of PCD, the delta psi(m) decrease and the ROS content increase. Addition of ascorbic acid (AsA) during the salt stress significantly decreased the percentage of protoplasts undergoing PCD and ROS levels but increased delta psi(m). Hydrogen peroxide effectively induced the appearance of PCD and caused an increase in ROS and a decrease in delta psi(m). Pre-treatment of protoplasts with CsA weakened the effects of H2O2. All these results suggest that the open state of PTP and ROS are necessary elements for salt stress-induced PCD in tobacco protoplasts. The open states of PTP and ROS could promote each other suggesting that ROS could lead to a self-amplifying process. This positive feedback loop may act as an all-or-nothing switch, which is in good accordance with the hypothesis that PTP is an important coordinator and executioner of PCD in both animals and plants.     

EDS1-mediated activation of autophagy regulates <Emphasis Type="Italic">Pst</Emphasis> DC3000 (<Emphasis Type="Italic">AvrRps4</Emphasis>)-induced programmed cell death in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Autophagy is a conserved intracellular process through which cytoplasmic components are degraded and recycled under stress conditions. In the innate immunity of higher plants, autophagy has either pro-survival or pro-death functions in pathogen-induced programmed cell death (PCD). In aged leaves, autophagy negatively regulates PCD by eliminating redundant salicylic acid. However, in young leaves, the specific pro-death mechanisms of autophagy and signaling pathways related to the autophagic process have not been elucidated. Here, we demonstrate that enhanced disease susceptibility 1 (EDS1) mediated the activation of autophagy and played a key role in the pro-death mechanism of autophagy during avirulent Pst DC3000 (AvrRps4) infection. The path through which autophagosomes enter the vacuole was blocked. Additionally, formation of the ATG12–ATG5 complex and the level of enzymatic activity associated with ATG8 cleavage decreased in eds1 mutants. The expression of EDS1 in atg5 mutants was also much lower than that in wild-type plants during pathogen-triggered PCD. These findings implied that EDS1 may regulate autophagy by affecting the activities of the two ubiquitin-like protein-conjugating pathways. Moreover, autophagy may regulate immunity-related PCD by affecting the expression of EDS1 in young plants. Our results provide important insights into the mechanisms of EDS1 in autophagy during infection with avirulent Pst DC3000 (AvrRps4) in Arabidopsis.     

阿拉伯半乳糖蛋白在被子植物有性生殖中的作用

阿拉伯半乳糖蛋白(arabinogalactan-proteins,AGPs)是一类主要分布在细胞表面和胞外基质中的糖蛋白.它们在植物的雄性器官(花粉、花粉管、精细胞)、雌性器官(柱头、花柱、子房)和胚胎(合子胚和体细胞胚)等组织和细胞中均有大量的表达.大量研究表明AGPs在被子植物有性生殖过程中起着非常重要的作用,既可能参与花粉管粘附、营养、传导或提供信号的作用,也可能参与受精过程中配子识别和受精后胚胎的发育与分化等过程.该文就其分子结构、特性以及在植物有性生殖过程中各种器官和组织内的表达和功能研究进展做了较为全面的概述.