Natural (non-pathogenic) death of the cortex of wheat and barley seminal roots,as evidenced by nuclear staining with acridine orange

Summary Nuclear staining with acridine orange was used to assess cell viability in the cortex of wheat and barley seminal roots from glasshouse and field experiments. Results from this method correlated well with nuclear assessments made in unstained or Feulgen-stained roots, and other evidence is presented to support the validity of the method. The pattern of root cortex death (RCD) was similar in wheat and barley and consistent over a wide range of conditions. Behind the extending root tip and zone of nucleate root hairs, nuclei disappeared progressively from the outer five (of six) cortical cell layers of the root axes, starting in the epidermis. Stainable nuclei remained in the sixth cell layer, next to the endodermis, and in most cell layers around the bases of root laterals and in a small region immediately below the grain. The onset of cell death was apparently related more to the age of a root region than to its distance behind the root tip, and it was not closely correlated with endodermal or stelar development assessed by staining with phloroglucinol/HCl. The rate of RCD was much faster in wheat than barley in both glasshouse and field conditions, and faster in some spring wheat cultivars than in others in the glasshouse. RCD occurred in sterile vermiculite and perlite and was not enhanced by the presence of soil microorganisms; nor was it enhanced in soil by the addition of the non-pathogenic fungal parasitesPhialophora radicicola var..graminicola orMicrodochium bolleyi. RCD is suggested to be endogenously controlled by the amount of photosynthate reaching the cortex. Its implications for growth of soil microorganisms and especially for growth and biological control of root-infecting fungi are discussed.     

EARLY SENESCENCE OF CORTICAL CELLS IN THE ROOTS OF CEREALS. HOW GOOD IS THE EVIDENCE?

There are numerous reports that cortical cells senesce in young, otherwise healthy main roots of cereals, including corn. These are based on apparent absence of nuclei in root segments or transverse sections after acridine-orange staining. Senescence is said to progress from the outer to the inner cortex basipetally from the root tip, except cells around branch bases where nuclei always stain. We studied axile roots of soil-grown cereals using various methods to detect nuclei primarily in longitudinal sections. No senescence marked by nuclear loss was found in healthy-looking intact cortices. Cortical cells of mature corn roots remained alive except where aerenchyma developed. No cortical death had occurred in barley, wheat, or oat seminal roots in 15-,17-, and 20-day-old plants, respectively, but cortical cells in older regions of seminal and nodal roots did collapse and slough off, but with no evidence for earlier loss of nuclei. Failure to detect acridine-orange-stained nuclei may not indicate that cells are senescent, and can be an artifact caused by sectioning method and wall impermeability. The effectiveness of other methods for evaluation of root cell vitality is discussed.     

Death of the cereal root cortex: its relevance to biological control of take-all

Cell death in the root cortex of cereals was assessed by an inability to detect nuclei, using acridine orangelfluorescence microscopy after fixation and mild acid hydrolysis. Seminal roots were scanned at x 100 magnification and their cortices were considered dead when nuclei were absent from all cell layers except the innermost one, adjacent to the endodermis; this cell layer remains alive long after the rest of the cortex has died. Cortical death of wheat and barley roots occurred in the absence of major pathogens. Cell death started behind the root hair zone of the main root axis, initially in the outermost cell layer of the cortex and then progressively inwards towards the endodermis; however, the cortex remained alive for a distance of c. 800 μm around emerging root laterals. The rate of cortical death was more rapid in wheat than in barley, both under field conditions and in the glasshouse at 20 °C. Thus, field-grown spring wheat (Sicca) showed 50% death of the root cortex in the top 6 cm of first seminal roots after 35 days (growth stage 1–2), whereas spring barley (Julia) showed 50% death of the root cortex after 67 days (growth stage 8). In the glasshouse, the top 9 cm of first seminal roots on 16-day plants showed 55% cortical death in wheat (Cappelle-Desprez) but only 2.5% cortical death in barley (Igri). The same rates of death were found in all subsequent seminal roots. The wheat root cortex died at the same rate in sterile and unsterile conditions, and at the same rate in the presence/absence of Phialophora radicicola Cain var. graminicola Deacon or Aureobasidium bolleyi (Sprague) von Arx. Hence, although P. radicicola and other soil microorganisms may benefit from root cortex death they do not exert biological control of take-all by enhancing or retarding the rate of this process. To study the effects of cortical death on take-all, Gaeumannomyces graminis (Sacc.) Arx & Olivier var. tritici Walker was point-inoculated at the tips and on older (5 and 15 day) regions of wheat seminal roots. After 17 days at 20 °C the fungus had grown to the same extent as runner-hyphae in all cases, but the severity of disease decreased with increasing age of the root cortex prior to inoculation; thus, G. graminis caused most extensive vascular discoloration and most intense vascular blockage in roots inoculated at their tips. Similar experiments on wheat and barley roots inoculated separately with P. radicicola and G. graminis suggest that at least three factors associated with cortical death influence infection by these fungi: (1) initially, cell death may enhance infection because nutrients are made available to the parasites and host resistance within the cortex is reduced; (2) weak parasites and soil saprophytes may colonise dead and dying cortices in competition with G. graminis and P. radicicola and thereby reduce infection by these fungi; (3) changes in the endodermis and adjacent cell layers may be associated with cortical death and may retard invasion of the stele. Future work will seek to establish the relative importance of these factors and extend this study to other cereal host-fungus combinations.     

Root Colonization by Bipolaris sorokiniana in Different Cereals and Relations to Lesion Development and Natural Root Cortical Cell Death

A GUS-transformed strain of B. sorokiniana was used to study the relationship between fungal growth, lesion development and natural root cortical cell death (RCD) in roots of different cereals. Roots of 10-day-old seedlings, grown on filter paper, were inoculated with the fungus and at different time intervals lesion size and GUS-activity in the roots were determined. A significant, positive correlation was found between GUS-activity and ergosterol content in barley roots infected with this transformed strain and these results indicate that GUS can be used as a marker to study fungal growth in plant tissue. The fungus had a slower growth rate in resistant barley varieties, i.e., those producing smaller lesions, than in more susceptible varieties.
In wheat and triticale, the fungal growth was faster than in barley, despite the smaller and lighter coloured lesions in these species. This may be explained by the fact that wheat and triticale have a faster rate of root cortical senescence than barley. Presumably, dying cortex cells cannot respond to the fungal invasion by producing phenolic compounds that cause browning of the tissue. Among seven investigated cereal species, there was a positive correlation between the degree of RCD and fungal biomass increase in roots after inoculation.     

Earlier onset of DNA fragmentation in leaves of wheat compared to barley and rye

We have found extensive nucleosomal fragmentation of native DNA extracted from leaves of healthy cereal plants, as indicated by ladder patterns on agarose gels and TUNEL staining. The time of first appearance of fragmentation differed among cereals. Native DNA from the first leaf of 10-day-old plants formed a clear ladder pattern of multiples of 180 bp fragments in wheat and triticale but not in barley and oats. In one cultivar of rye a weak ladder pattern occurred but not in another. Freezing and thawing of samples before DNA extraction resulted in much more extensive DNA fragmentation in wheat but not in rye and barley, indicating that DNA-degrading enzymes are present in the cytoplasm of wheat, but not in barley and rye, at this stage. In barley, nucleosomal fragmentation was first detected in 25-day-old plants. These results indicate that programmed cell death takes place in developing leaves of young cereal plants, but that the time of onset differs among cereal species.     

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.     

Developmental programmed cell death in primary roots of Sonoran Desert Cactaceae

Primary roots of two species of Sonoran Desert Cactaceae, Stenocereus gummosus and Pachycereus pringlei, have a determinate pattern of growth: meristematic cells divide only for a limited time and then differentiate. Detecting DNA fragmentation by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL), we have shown that programmed cell death (PCD) was not involved in meristem exhaustion. However, we found TUNEL-positive nuclei in the root hair and root cap cells of both species. Programmed cell death in root hair cells has not been previously reported, and the pattern of PCD events in the root cap differed from that described earlier. These data suggest that in the studied Cactaceae, PCD is involved in developmental adaptations related to the formation of a compact root system important for rapid seedling establishment in a desert environment. Participation of PCD in developmental loss of the root cap and in root hair renovation proposed in the current study implicates an evolutionary conserved link between PCD and differentiation processes in plants.     

Involvement of endonuclease G in nucleosomal DNA fragmentation under sustained endogenous oxidative stress

We have previously shown that inhibition of catalase and glutathione peroxidase activities by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS), respectively, in rat primary hepatocytes caused sustained endogenous oxidative stress and apoptotic cell death without caspase-3 activation. In this study, we investigated the mechanism of this apoptotic cell death in terms of nucleosomal DNA fragmentation. Treatment with ATZ+MS time-dependently increased the number of deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)-positive nuclei from 12 h, resulting in clear DNA laddering at 24 h. The deoxyribonuclease (DNase) inhibitor, aurintricarboxylic acid (ATA), completely inhibited nucleosomal DNA fragmentation but the pan-caspase inhibitor, z-VAD-fmk was without effects; furthermore, the cleavage of inhibitor of caspase-activated DNase was not detected, indicating the involvement of DNase(s) other than caspase-activated DNase. Considering that endonuclease G (EndoG) reportedly acts in a caspase-independent manner, we cloned rat EndoG cDNA for the first time. Recombinant EndoG alone digested plasmid DNA and induced nucleosomal DNA fragmentation in isolated hepatocyte nuclei. Recombinant EndoG activity was inhibited by ATA but not by hydrogen peroxide, even at 10 mm. ATZ+MS stimulation elicited decreases in mitochondrial membrane potential and EndoG translocation from mitochondria to nuclei. By applying RNA interference, the mRNA levels of EndoG were almost completely suppressed and the amount of EndoG protein was decreased to approximately half the level of untreated cells. Under these conditions, decreases in TUNEL-positive nuclei were significantly suppressed. These results indicate that EndoG is responsible, at least in part, for nucleosomal DNA fragmentation under endogenous oxidative stress conditions induced by ATZ+MS.     

Salt is Necessary for Nucleosomal DNA Fragmentation Induced by Caspase

For nucleosomal DNA fragmentation, one of the hallmarks of apoptosis, activated caspase, an apoptosis specific cysteine protease, is required to cleave ICAD/DFF45 that releases its complexed DNase, CAD/DFF40. The protein complex is located predominantly in the nuclei. Inconsistently, caspase alone cannot induce DNA fragmentation in the isolated nuclei without the addition of a cell extract or purified CAD/DFF40. In this study, however, it is demonstrated that under selected conditions with 50-75 mM: KCl or NaCl, caspase-3 and-7 can induce DNA fragmentation without the additional factor(s).     

Mechanisms of induction of apoptosis by anthraquinone anticancer drugs aclarubicin and mitoxantrone in comparison with doxorubicin: Relation to drug cytotoxicity and caspase-3 activation

We examined molecular events and morphological features associated with apoptosis induced by anthraquinone anticancer drugs aclarubicin, mitoxantrone and doxorubicin in two spontaneously immortalized cell lines (NIH 3T3 and B14) in relation to cytotoxicity of these drugs. The investigated cells showed similar sensitivity to aclarubicin but different sensitivity to doxorubicin and mitoxantrone: mitoxantrone was the most cytotoxic drug in both cell lines. All three drugs triggered both apoptosis and necrosis but none of these processes was positively correlated with their cytotoxicity. Apoptosis was the prevalent form of cell kill by aclarubicin, while doxorubicin and mitoxantrone induced mainly the necrotic mode of cell death. The extent and the timing of apoptosis were strongly dependent on the cell line, the type of the drug and its dose, and were mediated by caspase-3 activation. A significant increase in caspase-3 activity and the percentage of apoptotic cells, oligonucleosomal DNA fragmentation, chromatin condensation and formation of apoptotic bodies was observed predominantly in B14 cells. NIH 3T3 cells showed lesser changes and a lack of DNA fragmentation. Aclarubicin was the fastest acting drug, inducing DNA fragmentation 12 h earlier than doxorubicin, and 24 h earlier than mitoxantrone. Caspase-3 inhibitor Ac-DEVD-CHO did not show any significant effect on drug cytotoxicity and DNA nucleosomal fragmentation.     

Caspase-3 activity and carbonyl cyanide m-chlorophenylhydrazone-induced apoptosis in HL-60

The role of caspase proteases in carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced apoptosis of human promyelocytic HL-60 cells was examined. Treatment of HL-60 cells with micromolar concentrations of CCCP resulted in cell death, with typical apoptotic features such as chromatin condensation, formation of apoptotic bodies, nucleosomal fragmentation of DNA and a distinct increase in caspase-3 activity. The results, however, indicated that full caspase-3 inhibition by the selective inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone (Z-DEVD-FMK) did not prevent cell death, nor did it affect the manifestation of apoptotic hallmarks, including apoptotic bodies formation and nucleosomal DNA fragmentation. The only distinct effect that Z-DEVD-FMK exhibited was to retard the disruption of the plasma membrane. We therefore assume that caspase-3 activity itself is not essential for the manifestation of apoptotic features mentioned above. Similarly, the pan-specific caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD-FMK) did not prevent cell death. On the contrary, Z-VAD-FMK completely prevented DNA cleavage and apoptotic body formation, but it failed to completely counteract chromatin condensation. Thus, in the presence of Z-VAD-FMK, application of CCCP concentrations that otherwise induced apoptosis, resulted in the appearance of two morphologically different groups of dead cells with intact DNA. The first group included cells with necrotic-like nuclear morphology, and therefore could be taken as being "truly" necrotic in nature, because they had intact DNA. The cells of the second group formed small single-spherical nuclei with condensed chromatin. In spite of having intact DNA, they could not be taken as "truly" necrotic cells. It is evident that in the experimental system, caspase proteases play an essential role in the formation of apoptotic bodies and in the cleavage of nucleosomal DNA, but not in the condensation of chromatin. Therefore, it is likely that the choice between cell death modalities is not solely a matter of the caspase proteases present.     

Facile induction of apoptosis into plant cells associated with temperature-sensitive lethality shown on interspecific hybrid from the cross Nicotiana suaveolens x N. tabacum

Two lines of suspension culture cells were obtained from a hybrid seedling of Nicotiana suaveolens Lehm. x N. tabacum L. cv. Hicks-2 expressing temperature-sensitive lethality. One of them (LH line) was inducible cell death in accordance with the lethality at 28 degrees C but not under high-temperature conditions (36 degrees C). Another one (SH line) lost the lethality and survived at 28 degrees C. The cells of LH line showed apoptotic changes when they were cultured at 28 degrees C. Fragmentation of nuclei was correlated with the lethality in the cells, as confirmed by fluorimetry of the nuclear DNA using laser scanning cytometry. Agarose gel analysis of DNA extracted from the cells expressing the lethality revealed a specific ladder pattern suggesting nucleosomal fragmentation that is one of the biochemical characteristics of apoptosis. From these facts, we confirmed that the process of cell death leading to hybrid lethality in the cells is certainly apoptosis. Hybrid cells were used in the experiments to estimate the point of no return in temperature-sensitive lethality and to examine the influence of cation in DNA fragmentation during apoptosis. The utility of hybrid cells as an experimental system for studies of hybrid lethality and apoptosis in plants was confirmed.     

The cellular apoptosis of murine BW5147 thymoma during cold shock]

The mode of T-lymphoma cell death induced by cold shock was studied. The rewarming of cells at 37 degrees C following a brief period of cold (0 degrees C) resulted in internucleosomal DNA fragmentation. The cells underwent cold shock-mediated apoptosis only at a reduced (2%) serum concentration. The apoptosis was not blocked by macromolecular synthesis inhibitors such as cycloheximide and antinomycin D, or by Quin-2. EGTA per se was responsible for the initiation of cell death. Colchicine also induced internucleosomal fragmentation of DNA. Our findings suggest that cold shock induced apoptosis is associated with low temperature mediated disruption of microtubules. The role of Ca2+ and growth factors in cold shock induced cell death is discussed.     

Effect of Chilling on DNA Endoreplication in Root Cortex Cells and Root Hairs of Soybean Seedlings

Relative nuclear DNA contents in cortex parenchyma cells in root segments of 3- and 7-d-old soybean seedlings grown at 25 °C and in plants grown for 3 d at 25 °C, and then for 4 d at 10 °C, were determined with cytophotometry. Measurements revealed that in each variant the cortex cell nuclei with DNA content between 2C and 8C were in all the examined segments and nuclei with 8C – 16C DNA appeared in higher parts of roots. However, in chilled plant cells the number of 8C – 16C DNA nuclei was very low. Therefore, chilling inhibited endoreplication in comparison with plants grown at 25 °C for 7 d, and even reduced endopolyploidy level as compared to the initial seedlings, i.e. 3-d-old plants. DNA contents in root hairs grown at 25 °C (control) and in root hairs emerged at 10 °C were also determined. In controls 4C – 8C DNA nuclei predominated while in chilled plants an additional population of 2C – 4C DNA appeared. Thus a reduction of DNA synthesis was brought about by low temperature. The occurrence of an intermediate DNA contents besides those with full endoreplication cycles suggests the possibility of differential DNA replication. This suggestion seems to be supported by the lack of ³H-thymidine incorporation into root hair nuclei at the examined developmental stage both in control and chilled root hairs. The same number, but larger, chromocentric lumps in polyploid cortex cell nuclei of higher root zones, in comparison to meristematic nuclei, suggests that endoreduplication process occurred. This revised version was published online in July 2006 with corrections to the Cover Date.     

Apoptotic nuclear morphological change without DNA fragmentation.

Apoptosis is characterized morphologically by condensation and fragmentation of nuclei and cells and biochemically by fragmentation of chromosomal DNA into nucleosomal units [1]. CAD, also known as CPAN or DFF-40, is a DNase that can be activated by caspases [2] [3] [4] [5] [6]. CAD is complexed with its inhibitor, ICAD, in growing, non-apoptotic cells [2] [7]. Caspases that are activated by apoptotic stimuli [8] cleave ICAD. CAD, thus released from ICAD, digests chromosomal DNA into nucleosomal units [2] [3]. Here, we examine whether nuclear morphological changes induced by apoptotic stimuli are caused by the degradation of chromosomal DNA. Human T-cell lymphoma Jurkat cells, as well as their transformants expressing caspase-resistant ICAD, were treated with staurosporine. The chromosomal DNA in Jurkat cells underwent fragmentation into nucleosomal units, which was preceded by large-scale chromatin fragmentation (50-200 kb). The chromosomal DNA in cells expressing caspase-resistant ICAD remained intact after treatment with staurosporine but their chromatin condensed as found in parental Jurkat cells. These results indicate that large-scale chromatin fragmentation and nucleosomal DNA fragmentation are caused by an ICAD-inhibitable DNase, most probably CAD, whereas chromatin condensation during apoptosis is controlled, at least in part, independently from the degradation of chromosomal DNA.     

Apoptotic and non-apoptotic cell death in hormone-dependent glands

The proliferation of cells and cell death are involved in the maintenance of appropriate tissue homeostasis. In the present study, two different mechanisms of cell death were identified in the prostate and pituitary glands when morphological data, fragmentation of DNA, and TUNEL labelling of apoptotic nuclei were compared. Typical cell death by apoptosis was identified by morphological and molecular approaches in the prostate after orchidectomy. By contrast, neither DNA fragmentation nor TUNEL labelling were found in dead cells occurring in the pituitary gland after interruption of lactation. Regressing lactotrophs were characterised by condensation and disruption of the cytoplasmic matrix, but preserved intact nuclei until advanced stages of regression. Degenerating “dark” cells comparable to those described in the pituitary were also seen coexisting with typical apoptosis in the prostate epithelial lining of orchidectomised rats. Both forms of cell death could be clearly differentiated, because dark cells suffer severe alterations of cytoplasmic organelles while maintaining the integrity of the nucleus. In contrast, apoptotic cells present well-preserved cytoplasmic organelles, but grossly disrupted nuclei with fragmentation and condensation of chromatin.     

Comparison of rates of natural senescence of the root cortex of wheat (with and without mildew infection), barley,oats and rye

Summary In comparative tests in a glasshouse, the cortex of oat and rye roots senesced more slowly than the cortex of wheat and barley roots. Of the cereals tested, wheat showed the most rapid rate of root cortical senescence, and the rate was unaffected by inoculation of leaves withErysiphe graminis. The results are discussed in relation to infection by root pathogens.     

Apoptotic DNA fragmentation

Degradation of nuclear DNA into nucleosomal units is one of the hallmarks of apoptotic cell death. It occurs in response to various apoptotic stimuli in a wide variety of cell types. Molecular characterization of this process identified a specific DNase (CAD, caspase-activated DNase) that cleaves chromosomal DNA in a caspase-dependent manner. CAD is synthesized with the help of ICAD (inhibitor of CAD), which works as a specific chaperone for CAD and is found complexed with ICAD in proliferating cells. When cells are induced to undergo apoptosis, caspases-in particular caspase 3-cleave ICAD to dissociate the CAD:ICAD complex, allowing CAD to cleave chromosomal DNA. Cells that lack ICAD or that express caspase-resistant mutant ICAD thus do not show DNA fragmentation during apoptosis, although they do exhibit some other features of apoptosis and die. In this review, the molecular mechanism of and the physiological roles played by apoptotic DNA fragmentation will be discussed.