Kinetin induces cell death in root cortex cells of Vicia faba ssp. minor seedlings

The double fluorescence staining with acridine orange and ethidium bromide (AO/EB) revealed that treatment of Vicia faba ssp. minor seedlings with kinetin-induced programmed cell death (PCD) in root cortex cells. Kinetin-induced cell death reflected by the morphological changes of nuclei including their invagination, volume increase, chromatin condensation and degradation as well as formation of micronuclei showed by AO/EB and 4,6-diamidino-2-phenylindol staining was accompanied by changes including increase in conductivity of cell electrolytes secreted to culture media, decrease in the number of the G1- and G2-phase cells and appearance of fraction of hypoploid cells as the effect of DNA degradation without ladder formation. Decrease in the number of mitochondria and in the activity of cellular dehydrogenases, production of reactive oxygen species (ROS), appearance of small and then large lytic vacuoles and increase in the amount of cytosolic calcium ions were also observed. The PCD was also manifested by increased width and weight of apical fragments of roots as well as decreased length of cortex cells which led to shortening of the whole roots. The kinetin-induced PCD process was almost completely inhibited by adenine, an inhibitor of phosphoribosyl transferase, and mannitol, an inhibitor of ROS production. These cell-death hallmarks and pathway of this process suggested that the induction of kinetin-specific vacuolar type of death, expressed itself with similar intensity on both morphological and metabolic levels, was a transient protecting whole roots and whole seedlings against elimination.     

Oligochitosan induces programmed cell death in tobacco suspension cells

Oligochitosan has been proved to trigger plant cell death. To gain some insights into the mechanisms of oligochitosan-induced cell death, the nature of oligochitosan-induced cell death and the role of calcium (Ca²⁺), nitric oxide (NO) and hydrogen peroxide (H₂O₂) were studied in tobacco suspension cells. Oligochitosan-induced cell death occurred in cytoplasmic shrinkage, phosphatidylserine externalization, chromatin condensation, TUNEL-positive nuclei, cytochrome c release and induction of programmed cell death (PCD)-related gene hsr203J, suggesting the activation of PCD pathway. Pretreatment cells with cyclosporin A, resulted in reducing oligochitosan-induced cytochrome c release and cell death, indicating oligochitosan-induced PCD was mediated by cytochrome c. In the early stage, cells undergoing PCD showed an immediate burst in free cytosolic Ca²⁺ ([Ca²⁺]_cyt) elevation, NO and H₂O₂ production. Further study showed that these three signals were involved in oligochitosan-induced PCD, while Ca²⁺ and NO played a negative role in this process by modulating cytochrome c release.     

Systemic PCD occurs in TMV-tomato interaction

In hypersensitive response (HR), programmed cell death (PCD) is reported as a powerful defense mechanism in plant immune responses to pathogen. However, little is known about the PCD in systemic acquired resistance (SAR). Using tobacco mosaic virus (TMV) to infect the tomato (Lycopersicon esculentum cv. Jiafen 16) we found that localized TMV-infection could induce cell death in the uninoculated parts of the tomatoes, where the enzyme-linked immunosorbent assay (ELISA) showed no spreading virus. The biological and molecular characterization of this cell death was shown as following: chromatin condensed and formed peripheral conglomeration in nuclei; cell nucleus were TUNEL positive labeled; genomic DNA was fragmented and showed DNA laddering; mitochondria and chloroplast were disrupted; tonoplast and plasma membrane were shrunk and degradated. These results suggested that with an absence of TMV spread, the local TMV-infection on certain tomato leaves could induce systemic PCD in the root-tips, stem-apices and uninoculated leaves. The systemic PCD has various initiation and synchronization in such tissues and is distinct in inducement and exhibition from HR-PCD and SAR.     

Time-course of programmed cell death during leaf senescence in <Emphasis Type="Italic">Eucommia ulmoides</Emphasis>

Leaves of Eucommia ulmoides Oliv. harvested between April to November were examined for programmed cell death (PCD) during growth and senescence. Leaves developed in April, becoming fully expanded in late May, remaining unchanged until November when they started to dehisce. Falling leaves retained a green color. Our results showed that (1) mesophyll cells gradually reduced their nuclei from September to November, (2) positive TUNEL signals appeared on the nuclei from August, (3) ladder-like DNA fragmentation occurred in September and October, and (4) a 20-kDa Ca²⁺-dependent DNase appeared in these same months. In fallen leaves, intact mesophyll cell nuclei could not be detected, but a few cells around the vascular bundle had nuclei. Therefore, (1) programmed cell death (PCD) of leaf cells occurred in the leaves of E. ulmoides, (2) the progress of mesophyll cell PCD lasted for more than 2 months, and (3) PCD of leaf cells was asynchronous in natural senescing leaves. Electronic Publication     

Fusaric acid induction of programmed cell death modulated through nitric oxide signalling in tobacco suspension cells

Fusaric acid (FA) is a nonhost-selective toxin mainly produced by Fusarium oxysporum, the causal agent of plant wilt diseases. We demonstrate that FA can induce programmed cell death (PCD) in tobacco suspension cells and the FA-induced PCD is modulated by nitric oxide (NO) signalling. Cells undergoing cell death induced by FA treatment exhibited typical characteristics of PCD including cytoplasmic shrinkage, chromatin condensation, DNA fragmentation, membrane plasmolysis, and formation of small cytoplasmic vacuoles. In addition, caspase-3-like activity was activated upon the FA treatment. The process of FA-induced PCD was accompanied by a rapid accumulation of NO in a FA dose-dependent manner. Pre-treatment of cells with NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or NO synthase inhibitor N ^G-monomethyl-arginine monoacetate (L-NMMA) significantly reduced the rate of FA-induced cell death. Furthermore, the caspase-3-like activity and the expression of PAL and Hsr203J genes were alleviated by application of cPTIO or L-NMMA to FA-treated tobacco cells. This indicates that NO is an important factor involved in the FA-induced PCD. Our results also show that pre-treatment of tobacco cells with a caspase-3-specific inhibitor, Ac-DEVD-CHO, can reduce the rate of FA-induced cell death. These results demonstrate that the FA-induced cell death is a PCD and is modulated by NO signalling through caspase-3-like activation.     

Systemic PCD occurs in TMV-tomato interaction

In hypersensitive response (HR), programmed cell death (PCD) is reported as a powerful defense mechanism in plant immune responses to pathogen. However, little is known about the PCD in sys-temic acquired resistance (SAR). Using tobacco mosaic virus (TMV) to infect the tomato (Lycopersicon esculentum cv. Jiafen 16) we found that localized TMV-infection could induce cell death in the uninoculated parts of the tomatoes, where the enzyme-linked immunosorbent assay (ELISA) showed no spreading virus. The biological and molecular characterization of this cell death was shown as fol-lowing: chromatin condensed and formed peripheral conglomeration in nuclei; cell nucleus were TUNEL positive labeled; genomic DNA was fragmented and showed DNA laddering; mitochondria and chloroplast were disrupted; tonoplast and plasma membrane were shrunk and degradated. These re-sults suggested that with an absence of TMV spread, the local TMV-infection on certain tomato leaves could induce systemic PCD in the root-tips, stem-apices and uninoculated leaves. The systemic PCD has various initiation and synchronization in such tissues and is distinct in inducement and exhibition from HR-PCD and SAR.     

Optimization and quality assessment of adventitious roots culture in Panax quinquefolium L.

In this study, adventitious roots of Panax quinquefolium L. have been successfully established. The highest induction rate of roots was obtained in MS medium containing 3 mg L^?1 IBA after 4 weeks of culture. The culture conditions of adventitious root were optimized and evaluated with response surface methodology. The best culture conditions for root growth seemed to be 0.75 salt strength MS medium, 4.70 g L^?1 inoculum size and 40 days of culture. The active component contents of adventitious roots were compared with those of native roots. The total saponins content was found to be 16.28 mg g^?1 in native root and 4.64 mg g^?1 in adventitious root. The polysaccharide content of the adventitious root was 1.5 times higher than that in the native P. quinquefolium (30.54 vs. 20.28 mg g^?1).     

Microcystin-LR induces chromatin alterations and modulates neutral single-strand-preferring nuclease activity in Phragmites australis

Microcystin-LR (MCY-LR), a toxin produced mainly by freshwater cyanobacteria, is a potent inhibitor of type 1 and 2A protein phosphatases. As such, it induces biochemical, cellular and tissue alterations in vascular plants, including cell death. The aim of this study was the analysis of MCY-LR induced changes in the activity of single-strand preferring nuclease (SSP nuclease) isoenzymes that are possibly involved in programmed cell death (PCD) of Phragmites australis (common reed, an aquatic macrophyte) cells. We analyzed both single-stranded DNA (ssDNase) and double-stranded DNA (dsDNase) cleaving activities. Activity gels revealed a number of seven isoenzymes named bands A-G in control reed shoots and roots. Their activity was organ- and age-dependent. We stained nuclei of root tip meristematic cells and found total and marginal chromatin condensations at relatively short-term (2-10 days) cyanotoxin exposure. At 10-20 days of cyanotoxin treatment, the number of cells with condensed chromatin decreased, which coincided with the occurrence of necrotic cell death. In parallel, overall ssDNase activity increased in the short term (five days) and gradually decreased at 10-20 days of MCY-LR treatment. In this context, the most important changes occurred for isoenzyme G of 28-32 kDa in roots and isoenzyme F of 35-38 kDa in shoots. dsDNase activity of isoenzyme E was decreased by MCY-LR in shoots, but increased in roots at 10 days of exposure. We conclude that the early induction of chromatin condensation and increase of SSP nuclease activities is related to PCD that will lead to necrosis with the cease of all cellular activities, including a decrease in nuclease activity.     

Study on programmed cell death and dynamic changes of starch accumulation in pericarp cells of Triticum aestivum L.*

Features of programmed cell death (PCD) and dynamic changes of starch accumulation in developing pericarp cells of wheat (Triticum aestivum L.) were observed and analyzed by periodic acid–Schiff/toluidine blue O double staining, fluorescence staining, terminal deoxynucleotidyl transferase-mediated fluorescein deoxyuridine triphosphate nick-end labeling (TUNEL) and transmission electron microscopy. The results showed that cellular organelles were orderly disintegrated. TUNEL-positive nuclei were detected at 0 day after flowering (DAF), whereas nuclei showed significant features of degradation at 2 DAF, such as chromatin condensation, nuclei condensation, and nuclei deformation. Then, heterochromatin gradually disappeared and the cellular nucleus was completely degraded. The mitochondria degradation and vacuolation also were detected at 15 DAF. These results indicated that the development of pericarp cells was a typical process of PCD. However, the PCD in pericarp cells had their own characteristics: PCD started early and lasted for a considerable time. In the delayed process of PCD, starch granules were synthesized, deposited, and degraded temporarily in amyloplasts or chloroplasts. The delay of PCD in pericarp cells may be due to sufficient photosynthetic assimilates and energy supply. Besides, normal mitochondria were required for pericarp cells to survive. Pericarp cells contained only compound starch granules. Starch was massively synthesized from 0 to 11 DAF, but it was rapidly degraded after 11 DAF. Therefore, apart from protection, pericarp cells played essential roles in starch synthesis, storage, and degradation, as well as nutrient transportation.     

Determination of ACC-induced cell-programmed death in roots of Vicia faba ssp. minor seedlings by acridine orange and ethidium bromide staining

Fluorescence staining with acridine orange (AO) and ethidium bromide (EB) showed that nuclei of cortex root cells of 1-aminocyclopropane-1-carboxylic acid (ACC)-treated Vicia faba ssp. minor seedlings differed in color. Measurement of resultant fluorescence intensity (RFI) showed that it increased when the color of nuclear chromatin was changed from green to red, indicating that EB moved to the nuclei via the cell membrane which lost its integrity and stained nuclei red. AO/EB staining showed that changes in color of the nuclear chromatin were accompanied by DNA condensation, nuclei fragmentation, and chromatin degradation which were also shown after 4,6-diamidino-2-phenylindol staining. These results indicate that ACC induced programmed cell death. The increasing values of RFI together with the corresponding morphological changes of nuclear chromatin were the basis to prepare the standard curve; cells with green unchanged nuclear chromatin were alive while those with dark orange and bright red nuclei were dead. The cells with nuclei with green–yellow, yellow–orange, and bright orange chromatin with or without their condensation and fragmentation chromatin were dying. The prepared curve has became the basis to draw up the digital method for detection and determination of the number of living, dying, and dead cells in an in planta system and revealed that ACC induced death in about 20% of root cortex cells. This process was accompanied by increase in ion leakage, shortening of cells and whole roots, as well as by increase in weight and width of the apical part of roots and appearance of few aerenchymatic spaces while not by internucleosomal DNA degradation.     

Programmed cell death in plants: Protective effect of mitochondrial-targeted quinones

Ubiquinone or plastoquinone covalently linked to synthetic decyltriphenylphosphonium (DTPP⁺) or rhodamine cations prevent programmed cell death (PCD) in pea leaf epidermis induced by chitosan or CN⁻. PCD was monitored by recording the destruction of cell nuclei. CN⁻ induced the destruction of nuclei in both epidermal cells (EC) and guard cells (GC), whereas chitosan destroyed nuclei in EC not in GC. The half-maximum concentrations for the protective effects of the quinone derivatives were within the pico- and nanomolar range. The protective effect of the quinones was removed by a protonophoric uncoupler and reduced by tetraphenylphosphonium cations. CN⁻-Induced PCD was accelerated by the tested quinone derivatives at concentrations above 10⁻⁸–10⁻⁷ M. Unlike plastoquinone linked to the rhodamine cation (SkQR1), DTPP⁺ derivatives of quinones suppressed menadione-induced H₂O₂ generation in the cells. The CN⁻-induced destruction of GC nuclei was prevented by DTPP⁺ derivatives in the dark not in the light. SkQR1 inhibited this process both in the dark and in the light, and its effect in the light was similar to that of rhodamine 6G. The data on the protective effect of cationic quinone derivatives indicate that mitochondria are involved in PCD in plants.     

Programmed cell death during floral nectary senescence in Ipomoea purpurea

The nectaries of Ipomoea purpurea wilt in the late flowering period. The senescence process of nectaries is frequently associated with cell lysis. In this paper, various techniques were used to investigate whether programmed cell death (PCD) was involved in the senescence process of nectaries in I. purpurea. Ultrastructural studies showed that nectary cells began to undergo structural distortion, chromatin condensation, mitochondrial membrane degradation, and vacuolar-membrane dissolution and rupture after bloom. 4′,6-Diamidino-2-phenylindole (DAPI) and terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine-5′-triphosphate (dUTP) nick end-labeling (TUNEL) assay showed that nectary cell nuclear DNA began to degrade during the budding stage, and disappeared in the fruiting stage. DNA gel electrophoresis showed that degradation of DNA was random. Together, these results suggest that PCD participate in the senescence of the nectary in I. purpurea. PCD began during the budding period, followed by significant changes in nectary morphology and structure during the flowering period. During the fruiting stage, the PCD process is complete and the nectary degrades.     

Adding Zn2+ induces DNA fragmentation and cell condensation in cultured human Chang liver cells

Zinc (Zn) is a trace element in human cells and regarded as an essential nutrient with established deficiency states affecting multiple organs in the body. However, it has been reported that Zn uptake is associated with some serious harmful effects, such as inhibition of DNA synthesis and enhanced toxicity from reactive oxygen species. We have previously shown that in vivo administration of Zn²⁺ in C57/6J mice induces weight loss and massive hair loss where the normal course hair becomes replaced by fine vello hair, simulating the side effects from cancer chemotherapy where oxidative free radical damage is implicated in association with DNA fragmentation and programmed cell death (PCD). Here, in vitro flow cytometric studies on human Chang liver showed Zn²⁺ causing cell condensation with DNA fragmentation that occurred in a dose-dependent manner, an effect replicated by micrococcal nuclease digestion. Specific terminal deoxynucleotidyl transferase- (TdT) mediated labeling of 3′-OH ends of DNA nicks corroborated the flow cytometric profiles of propidium iodide-DNA binding where degradation of both 2 and 4N genomic DNA resulted in a solitary 1N peak presentation. DNA degradation concomitant with cell condensation is seen as an estabilished hallmark of PCD. We further showed that Zn²⁺ could enhance the generation of hydroxyl free radicals (OH^?) by the transition metal vanadium. Glutathione, the cell's main reducing agent, underwent corresponding reduction. The results suggested that Zn supplementation could induce features resembling PCD.     

Effects of waterlogging on amyloplasts and programmed cell death in endosperm cells of Triticum aestivum L.

The effects of waterlogging on amyloplasts and programmed cell death (PCD) in endosperm cells in Chinese wheat (Triticum aestivum L.; cv: Hua mai 8) are here discussed. Four water treatments were established from anthesis to maturity: they were 3 days of waterlogging treatment (DWT), 7 DWT, 12 DWT, and moderate water supply (the control). Lugol staining and scanning electron microscopy showed decreases in the number of amyloplasts and partially filled circular cavities under the waterlogging treatments. These resulted in serious deformities in the endosperm cells. Evans blue staining analysis and terminal deoxynucleotidyl transferase-mediated fluorescein deoxyuridine triphosphate nick-end labeling assays indicated that the PCD progression of endosperm cells occurred earlier under waterlogging treatments than in the control, so did the internucleosomal DNA fragmentation, which accompanies PCD in endosperm cells. Electron transmission microscopy analysis showed similar results. Under waterlogging treatments, the following PCD characteristics appeared earlier and were more pronounced than in normal endosperm cells: chromatin condensation, degradation of the nuclear envelope, swelling, and degradation of the mitochondrial cristae. Our study concluded that under waterlogging conditions, the number of amyloplasts tended to decrease and PCD was likely to appear ahead of time.     

The release of cytochrome c and the regulation of the programmed cell death progress in the endosperm of winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> 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.     

Immunohistological localization and quantification of IAA in studies of root growth regulation

The content and distribution of auxins were studied in gravistimulated roots of maize (Zea mays L.) and primary roots of 7-day-old wheat (Triticum durum Desf.) seedlings, which branching was enhanced by excision of adventitious roots. IAA localization was observed immunohistochemically, using specific anti-IAA antibody in combination with second (anti-species) antibody labeled with colloidal gold. Differences in the IAA content (staining intensity) were found between upper and lower parts of gravistimulated maize roots. We also observed IAA accumulation in the primary wheat root after adventitious root excision; the cells of lateral root primordia were characterized by more intense IAA staining. The role of auxin redistribution in plants for lateral root initiation and development is discussed.     

A cascade signal pathway occurs in self-incompatibility of Pyrus pyrifolia

Pear (Pyrus pyrifolia L.) possesses an S-RNase-based gametophytic self-incompatibility (GSI) system and S-RNase, the self-incompatibility (SI) determinant in the pistil, has also been implicated in the rejection of self-pollen and genetically identical pollen. We have demonstrated that S-RNase depolymerises actin cytoskeleton, triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube, which indicates programmed cell death (PCD) may occur in SI response of Pyrus pyrifolia. Recently, we have identified that S-RNase specifically disrupted tip-localized reactive oxygen species (ROS) of incompatible pollen tube via arrest of ROS formation in mitochondria and cell walls in Pyrus pyrifolia. Furthermore, tip-localized ROS disruption not only decreased the Ca²⁺ current and depolymerised the actin cytoskeleton, but it also induced nuclear DNA degradation in the pollen tube. The results mentioned above indicate that a cascade signal pathway may occur in SI of Pyrus pyrifolia and PCD is used to terminate the incompatible pollen tubes growth. In this addendum, we review the cascade signal pathway of Pyrus pyrifolia SI.Key words: S-RNase, programmed cell death, reactive oxygen species, actin cytoskeleton, Ca²⁺ current, nuclear DNA