The opening of mitochondrial permeability transition pore (mPTP) and the inhibition of electron transfer chain (ETC) induce mitophagy in wheat roots under waterlogging stress.

Mitochondria are crucial for the regulation of intracellular energy metabolism, biosynthesis, and cell survival. And studies have demonstrated the role of mitochondria in oxidative stress-induced autophagy in plants. Previous studies found that waterlogging stress can induce the opening of mitochondrial permeability transition pore (mPTP) and the release of cytochrome c in endosperm cells, which proved that mPTP plays an important role in the programmed cell death of endosperm cells under waterlogging stress. This study investigated the effects of the opening of mPTP and the inhibition of ETC on mitophagy in wheat roots under waterlogging stress. The results showed that autophagy related genes in the mitochondria of wheat root cells could respond to waterlogging stress; waterlogging stress led to the degradation of the characteristic proteins cytochrome c and COXII in the mitochondria of root cells. With the prolongation of waterlogging time, the protein degradation degree and the occurrence of mitophagy gradually increased. Under waterlogging stress, exogenous mPTP opening inhibitor CsA inhibited mitophagy in root cells and alleviated mitophagy induced by flooding stress, while exogenous mPTP opening inducer CCCP induced mitophagy in root cells; exogenous mPTP opening inducer CCCP induced mitophagy in root cells. The electron transfer chain inhibitor antimycin A induces mitophagy in wheat root cells and exacerbates mitochondrial degradation. In conclusion, waterlogging stress led to the degradation of mitochondrial characteristic proteins and the occurrence of mitophagy in wheat root cells, and the opening of mPTP and the inhibition of ETC induced the occurrence of mitophagy.

Short-term waterlogging-induced autophagy in root cells of wheat can inhibit programmed cell death.

Autophagy is a pathway for the degradation of cytoplasmic components in eukaryotes. In wheat, the mechanism by which autophagy regulates programmed cell death (PCD) is unknown. Here, we demonstrated that short-term waterlogging-induced autophagy inhibited PCD in root cells of wheat. The waterlogging-tolerant wheat cultivar Huamai 8 and the waterlogging-sensitive wheat cultivar Huamai 9 were used as experimental materials, and their roots were waterlogged for 0-48 h. Waterlogging stress increased the number of autophagic structures, the expression levels of autophagy-related genes (TaATG), and the occurrence of PCD in root cells. PCD manifested as morphological changes in the cell nucleus, significant enhancement of DNA laddering bands, and increases in caspase-like protease activity and the expression levels of metacaspase genes. The autophagy promoter rapamycin (RAPA) reduced PCD levels, whereas the autophagy inhibitor 3-methyladenine (3-MA) enhanced them. The expression levels of TaATG genes and the number of autophagic structures were lower in cortex cells than in stele cells, but the levels of PCD were higher in cortex cells. The number of autophagic structures was greater in Huamai 8 than in Huamai 9, but the levels of PCD were lower. In summary, our results showed that short-term waterlogging induced autophagy which could inhibit PCD. Mechanisms of response to waterlogging stress differed between cortex and stele cells and between two wheat cultivars of contrasting waterlogging tolerance.

Mutual regulation of ROS accumulation and cell autophagy in wheat roots under hypoxia stress.

Here, we explored the mutual regulation of radical oxygen species (ROS) and autophagy in wheat (Triticum aestivum L.) roots under hypoxia stress. We also analyzed differences between the responses of the stele and the cortex in the two wheat cultivars Huamai 8 (waterlogging-tolerant) and Huamai 9 (waterlogging-sensitive) to hypoxia stress. In situ detection and ultracytochemical localization analysis in wheat roots showed that hypoxia stress caused greater increases in ROS levels and the expression levels of alternative oxidase (AOX) and antioxidant enzymes in the stele than in the cortex. The analysis of exogenous ROS addition and the inhibition of its production revealed the pivotal roles played by ROS in autophagy. Moreover, transmission electron microscopy and qRT-PCR analysis indicated that the stele had a higher level of autophagy than the cortex and that the two wheat cultivars primarily differed in the type and number of autophagosomes. Additional research revealed that autophagy could remove excess ROS, as pre-treatment with the autophagy inhibitor 3-methyladenine increased ROS levels in roots and the addition of the autophagy inducer rapamycin reduced root ROS levels. In conclusion, hypoxia stress induced ROS accumulation in wheat roots where ROS acted as an autophagy signal. Furthermore, higher levels of autophagy and antioxidant enzyme expression in the stele facilitated the elimination of oxidative damage caused by excessive ROS and thereby increased cell survival; in the cortex, a large number of cells died and formed aerenchyma.

Effect of Waterlogging-Induced Autophagy on Programmed Cell Death in Arabidopsis Roots.

Autophagy, a highly conserved process in eukaryotes that involves vacuolar degradation of intracellular components and decomposition of damaged or toxic constituents, is induced by endogenous reactive oxygen species (ROS) accumulation, endoplasmic reticulum stress, and other factors. In plants, the role of autophagy in the induction of programmed cell death (PCD) is still unclear. Here, we show that ROS contribute to the regulation of PCD during waterlogging (which results in oxygen depletion) via autophagy. In wild-type roots, waterlogging induces the transcription of hypoxia-responsive genes and respiratory burst oxidase homolog (RBOH)-mediated ROS production. It also altered the transcription level of alternative oxidase1a and the activity level of antioxidant enzymes. Moreover, waterlogging increased the transcription levels of autophagy-related (ATG) genes and the number of autophagosomes. Autophagy first occurred in the root stele, and then autophagosomes appeared at other locations in the root. In rboh mutants, upregulation of autophagosomes was less pronounced than in the wild type upon waterlogging. However, the accumulation of ROS and the level of cell death in the roots of atg mutants were higher than those in the wild type after waterlogging. In conclusion, our results suggest that autophagy induced in Arabidopsis roots during waterlogging has an attenuating effect on PCD in the roots.

The release of cytochrome c and the regulation of the programmed cell death progress in the endosperm of winter wheat (Triticum aestivum L.) under waterlogging.

It has been shown in mammalian systems that the mitochondria can play a key role in the regulation of apoptosis by releasing intermembrane proteins (such as cytochrome c) into the cytosol. Cytochrome c released from the mitochondria to the cytoplasm activates proteolytic enzyme cascades, leading to specific nuclear DNA degradation and cell death. This pathway is considered to be one of the important regulatory mechanisms of apoptosis. Previous studies have shown that endosperm cell development in wheat undergoes specialized programmed cell death (PCD) and that waterlogging stress accelerates the PCD process; however, little is known regarding the associated molecular mechanism. In this study, changes in mitochondrial structure, the release of cytochrome c, and gene expression were studied in the endosperm cells of the wheat (Triticum aestivum L.) cultivar "huamai 8" during PCD under different waterlogging durations. The results showed that waterlogging aggravated the degradation of mitochondrial structure, increased the mitochondrial permeability transition (MPT), and decreased mitochondrial transmembrane potential (ΔΨm), resulting in the advancement of the endosperm PCD process. In situ localization and western blotting of cytochrome c indicated that with the development of the endosperm cell, cytochrome c was gradually released from the mitochondria to the cytoplasm, and waterlogging stress led to an advancement and increase in the release of cytochrome c. In addition, waterlogging stress resulted in the increased expression of the voltage-dependent anion channel (VDAC) and adenine nucleotide translocator (ANT), suggesting that the mitochondrial permeability transition pore (MPTP) may be involved in endosperm PCD under waterlogging stress. The MPTP inhibitor cyclosporine A effectively suppressed cell death and cytochrome c release during wheat endosperm PCD. Our results indicate that the mitochondria play important roles in the PCD of endosperm cells and that the increase in mitochondrial damage and corresponding release of cytochrome c may be one of the major causes of endosperm PCD advancement under waterlogging.

Reactive oxygen species regulate programmed cell death progress of endosperm in winter wheat (Triticum aestivum L.) under waterlogging.

Previous studies have proved that waterlogging stress accelerates the programmed cell death (PCD) progress of wheat endosperm cells. A highly waterlogging-tolerant wheat cultivar Hua 8 and a waterlogging susceptible wheat cultivar Hua 9 were treated with different waterlogging durations, and then, dynamic changes of reactive oxygen species (ROS), gene expressions, and activities of antioxidant enzymes in endosperm cells were detected. The accumulation of ROS increased considerably after 7 days of waterlogging treatment (7 DWT) and 12 DWT in both cultivars compared with control group (under non-waterlogged conditions), culminated at 12 DAF (days after flowering) and reduced hereafter. Waterlogging resulted in a great increase of H2O2 and O2 (-) in plasma membranes, cell walls, mitochondrias, and intercellular spaces with ultracytochemical localization. Moreover, the deformation and rupture of cytomembranes as well as the swelling and distortion of mitochondria were obvious. Under waterlogging treatment conditions, catalase (CAT) gene expression increased in endosperm of Hua 8 but activity decreased. In addition, Mn superoxide dismutase (MnSOD) gene expression and superoxide dismutase (SOD) activity increased. Compared with Hua 8, both CAT, MnSOD gene expressions and CAT, SOD activities decreased in Hua 9. Moreover, ascorbic acid and mannitol relieve the intensifying of PCD processes in Hua 8 endosperm cells induced by waterlogging. These results indicate that ROS have important roles in the PCD of endosperm cells, the changes both CAT, MnSOD gene expressions and CAT, SOD activities directly affected the accumulation of ROS in two different wheat cultivars under waterlogging, ultimately led to the PCD acceleration of endosperm.

Microautophagy involves programmed cell semi-death of sieve elements in developing caryopsis of Triticum aestivum L.

Differentiation of sieve elements (SEs) involves programmed cell semi-death, in which a small amount of organelles is retained. However, the mechanisms by which a large amount of SE cytoplasm is degraded and the specific proteases involved are not clear. In this study, we confirmed that the degradation of cytoplasm outside of the vacuole was mediated by microautophagy of the vacuole, and that the tonoplast selectively fused with the plasma membrane after most of the cytoplasm in the vacuoles was degraded. The integration of space enclosed a small amount of cytoplasm. Therefore, that fraction of the cytoplasm was preserved. At the same time, the cytosol was weakly acidic during membrane fusion because part of the tonoplast was ruptured. We also demonstrated that wheat aspartic protease (WAP1) and proteases including cathepsin B activity (PICA) were involved in programmed cell semi-death of SEs. PICA showed strongest activity before mass of the cytoplasm was degraded, which might contribute toward SE stability. We found that WAP1 mainly degraded the cytoplasm. Therefore, programmed cell semi-death of SEs might result from the joint action of vacuoles and multiple proteases.

Localization of BEN1-LIKE protein and nuclear degradation during development of metaphloem sieve elements in Triticum aestivum L.

Metaphloem sieve elements (MSEs) in the developing caryopsis of Triticum aestivum L. undergo a unique type of programmed cell death (PCD); cell organelles gradually degrade with the MSE differentiation while mature sieve elements keep active. This study focuses on locating BEN1-LIKE protein and nuclear degradation in differentiating MSEs of wheat. Transmission electron microscopy (TEM) showed that nuclei degraded in MSE development. First, the degradation started at 2-3 days after flowering (DAF). The degraded fragments were then swallowed by phagocytic vacuoles at 4 DAF. Finally, nuclei almost completely degraded at 5 DAF. We measured the BEN1-LIKE protein expression in differentiating MSEs. In situ hybridization showed that BEN1-LIKE mRNA was a more obvious hybridization signal at 3-4 DAF at the microscopic level. Immuno-electron microscopy further revealed that BEN1-LIKE protein was mainly localized in MSE nuclei. Furthermore, MSE differentiation was tested using a TSQ Zn2+ fluorescence probe which showed that the dynamic change of Zn2+ accumulation was similar to BEN1-LIKE protein expression. These results suggest that nucleus degradation in wheat MSEs is associated with BEN1-LIKE protein and that the expression of this protein may be regulated by Zn2+ accumulation variation.

Evidence of ceased programmed cell death in metaphloem sieve elements in the developing caryopsis of Triticum aestivum L.

Transmission electron microscopy (TEM) and fluorescence microscopy studies revealed that the metaphloem sieve elements (MSEs) in the ventral vascular bundle of the caryopses of developing wheat (Triticum aestivum L.) undergo a unique type of programmed cell death (PCD). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive nuclei were observed at 3 and 4 days after flowering (DAF). Transmission electron microscopy studies of differentiating MSEs revealed increased vacuolation, nuclear degeneration, chromatin condensation and localization to the periphery of the nucleus, and partly dilated perinuclear spaces, all typical characteristics of PCD in plant cells. In addition, vacuoles were disrupted at the last stages of differentiation. These results demonstrate that MSE differentiation is a unique type of PCD with highly selective autophagic processes, in which PCD ceases just prior to death. During this cessation of PCD, vacuoles and the endoplasmic reticulum appear to be associated with selective organelle digestion.