Bacterial lipopolysaccharides induce defense responses associated with programmed cell death in rice cells

PAMP (pathogen-associated molecular pattern) recognition plays an important role during the innate immune response in both plants and animals. Lipopolysaccharides (LPS) derived from Gram-negative bacteria are representative of typical PAMP molecules and have been reported to induce defense-related responses, including the suppression of the hypersensitive response, the expression of defense genes and systemic resistance in plants. However, the details regarding the precise molecular mechanisms underlying these cellular responses, such as the molecular machinery involved in the perception and transduction of LPS molecules, remain largely unknown. Furthermore, the biological activities of LPS on plants have so far been reported only in dicots and no information is thus available regarding their functions in monocots. In our current study, we report that LPS preparations for various becteria, including plant pathogens and non-pathogens, can induce defense responses in rice cells, including reactive oxygen generation and defense gene expression. In addition, global analysis of gene expression induced by two PAMPs, LPS and chitin oligosaccharide, also reveals a close correlation between the gene responses induced by these factors. This indicates that there is a convergence of signaling cascades downstream of their corresponding receptors. Furthermore, we show that the defense responses induced by LPS in the rice cells are associated with programmed cell death (PCD), which is a finding that has not been previously reported for the functional role of these molecules in plant cells. Interestingly, PCD induction by the LPS was not detected in cultured Arabidopsis thaliana cells.     

The PET1-CMS mitochondrial mutation in sunflower is associated with premature programmed cell death and cytochrome c release

In mammals, mitochondria have been shown to play a key intermediary role in apoptosis, a morphologically distinct form of programmed cell death (PCD), for example, through the release of cytochrome c, which activates a proteolytic enzyme cascade, resulting in specific nuclear DNA degradation and cell death. In plants, PCD is a feature of normal development, including the penultimate stage of anther development, leading to dehiscence and pollen release. However, there is little evidence that plant mitochondria are involved in PCD. In a wide range of plant species, anther and/or pollen development is disrupted in a class of mutants termed CMS (for cytoplasmic male sterility), which is associated with mutations in the mitochondrial genome. On the basis of the manifestation of a number of morphological and biochemical markers of apoptosis, we have shown that the PET1-CMS cytoplasm in sunflower causes premature PCD of the tapetal cells, which then extends to other anther tissues. These features included cell condensation, oligonucleosomal cleavage of nuclear DNA, separation of chromatin into delineated masses, and initial persistence of mitochondria. In addition, immunocytochemical analysis revealed that cytochrome c was released partially from the mitochondria into the cytosol of tapetal cells before the gross morphological changes associated with PCD. The decrease in cytochrome c content in mitochondria isolated from male sterile florets preceded a decrease in the integrity of the outer mitochondrial membrane and respiratory control ratio. Our data suggest that plant mitochondria, like mammalian mitochondria, play a key role in the induction of PCD. The tissue-specific nature of the CMS phenotype is discussed with regard to cellular respiratory demand and PCD during normal anther development.     

Formation of archegonium chamber is associated with nucellar-cell programmed cell death in Ginkgo biloba

Summary. The archegonium chamber in Ginkgo biloba L. is a pathway for spermatozoids swimming towards the archegonium for fertilization. The objective of this study was to elucidate the mechanism of archegonium chamber formation. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and DNA ladder demonstrated that the nucellar cell death, coordinated with the archegonium chamber formation, was a process of programmed cell death. Cytochemical localization of Ca²⁺ in these nucellar cells was determined by means of in situ precipitation with potassium pyroantimonate and electron microscopic visualization, in order to study the relation between Ca²⁺ and programmed cell death. The results showed an early uptake of the mitochondrial calcium particles in the nucellar cells undergoing programmed cell death. Together with other dynamic changes in Ca²⁺ subcellular distribution, this indicates that Ca²⁺ may play a role in the regulation of mitochondria-mediated programmed events in the nucellar cells. Correspondence and reprints: School of Pharmaceutical Sciences, Peking University, Beijing 100083, People’s Republic of China.     

光敏核不育水稻的温光反应研究

本文选用W_(6154)S、安农S-1和衡农S-13个水稻光敏核不育系做材料,采用分期播种的办法研究,提出了“临界低温”、“临界高温”和“过渡温度”的概念,求得W_(6154)S和安农S-1的临界低温为23℃,临界高温为25℃;衡农S-1的临界低温为23.5℃,临界高温为27℃。认为(1)不同的光敏核不育系的温光反应特性不同;(2)以“生态因子敏感雄性核不育水稻”这一名称代替“光敏核不育水稻”为宜;(3)在生产上可以应用。     

Calcium distribution in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice

Potassium antimonate was used to locate Ca²⁺ in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice (Oryza sativa L. japonica). During the development of fertile anthers, abundant calcium precipitates accumulated in the anther walls and on the surface of pollen grains and Ubish bodies at the late developmental stage of the microspore, but not in the cytoplasm of pollen grains. Following the accumulation of starch grains in pollen, calcium precipitates on pollen walls diminished and increased in parenchymatous cells of the connective tissue. In sterile anthers, calcium precipitates were abundant in the middle layer and endothecium, but not in the tapetum, as was found in fertile anthers. A special cell wall was observed between the tapetum and middle layer of sterile anthers that appeared to relate to distinctive calcium accumulation patterns and poor pollen wall formation in the loculi. The formation of different patterns of antimonate-induced calcium precipitates in the anthers of photoperiod-sensitive genic male-sterile rice indicates that anomalies in the distribution of calcium accumulation correlate with the failure of pollen development and pollen abortion. Received: 30 May 1997 / Accepted: 5 July 1997     

Molecular mapping of the reverse thermo-sensitive genic male-sterile gene (rtms1) in rice

TGMS (thermo-sensitive genic male-sterile) rice is widely used in hybrid rice production. Because of a specific temperature requirement, it can be used only in a narrow rice-growing zone in Asia. A newly discovered reverse thermo-sensitive genic male-sterile line, J207S, has an opposite phynotype compared to the normal TGMS lines. J207S is completely sterile when the temperature is lower than 31°C. Thus, it can be widely used in a larger area. Genetic analysis indicated that the sterility of J207S was controlled by a single recessive gene which was first named as rtms1. An F₂ population from the cross between J207S and E921 was developed and used for molecular mapping of the rtms1 gene. The AFLP (amplified fragment length polymorphism) technique, combined with BSA (bulked segregant analysis), was used to screen markers linked to the target gene, and eight polymorphic AFLP loci were identified. Co-segregating analysis using the F₂ population showed that two of them, Rev1 and Rev7, were closely linked to the target gene with a recombinant rate of 3.8% and 7.7%, respectively. Both Rev1 and Rev7 were found to be single-copy sequences through Southern analysis. Rev1 was subsequently mapped on chromosome 10 with a doubled-haploid mapping populations derived from the cross CT9993 × IR62266 available at Texas Tech University. RM222 and RG257 were linked to Rev1 at a distance of 11.8 cM and 4.6 cM, respectively. Additional SSR markers from the rice map of Cornell University, RFLP markers from the map of RGP in Japan and the map of Texas Tech University were selected from the region surrounding Rev1 on chromosome 10 to conduct the fine-mapping of the rtms1 gene. Presently, rtms1 was mapped between RM239 and RG257 with genetic distance of 3.6 cM and 4.0 cM, respectively. The most-closely linked AFLP marker, Rev1, 4.2 cM from the rtms1 gene, was sequenced and converted into a SCAR (sequence characterized amplified region) marker which could facilitate marker-assisted selection of the rtms1 gene. Received: 2 November 2000 / Accepted: 21 November 2000     

Identification of mRNAs associated with programmed cell death in immature thymocytes.

Programmed cell death is an essential cellular process that occurs in epithelial turnover, neural development, and regulation of cell populations of the immune system. Thymocytes undergo programmed cell death in response to several inductive stimuli, including exposure to glucocorticoids or radiation. This program can be blocked by inhibitors of RNA or protein synthesis; this implies that new proteins are required to execute the death programs. To search for possible death-associated mRNAs, we directionally cloned cDNA representing mRNA from control and dexamethasone-treated thymocytes. These libraries were used to produce ample amounts of DNA and RNA used in subtractive hybridization for the removal of sequences present in both control and induced cells. The remaining unhybridized sequences were selectively amplified by polymerase chain reaction and cloned to produce a library enriched for sequences expressed in death-induced cells. From this library we isolated cDNAs of death-associated mRNAs. One of these mRNAs, RP-8, appears within 1 h after exposure to gamma radiation, and a second mRNA, RP-2, is observed within 2 h. Both of these mRNAs accumulate during a period when a reference mRNA, actin, is declining. RP-2 and RP-8 are no longer detectable after 6 h postinduction, when apoptosis and mRNA degradation are evident in the culture. Sequence analysis of RP-8 cDNA indicates the presence of a zinc finger domain suggestive of a possible DNA regulatory role for the RP-8 protein. cDNA sequence results on RP-2 classify the corresponding protein as an integral membrane protein. We conclude that RP-2 and RP-8 are death-associated mRNAs that should be functionally evaluated in the context of the death process. As previously suggested, it may be that a family of death genes is activated by various stimuli depending on the type of cell, in a manner somewhat analogous to the induction of heat shock (stress) protein genes.     

Tagging and mapping the thermo-sensitive genic male-sterile gene in rice (Oryza sativa L.) with molecular markers

The thermo-sensititve genic male-sterile (TGMS) gene in rice can alter fertility in response to temperature and is useful in the two-line system of hybrid rice production. However, little is known about the TGMS gene at the molecular level. The objective of this study was to identify molecular markers tightly linked with the TGMS gene and to map the gene onto a specific rice chromosome. Bulked segregant analysis of an F₂ population from 5460s (a TGMS mutant line) x Hong Wan 52 was used to identify RAPD markers linked to the rice TGMS gene. Four hundred RAPD primers were screened for polymorphisms between the parents and between two bulks representing fertile and sterile plants; of these, 4 primers produced polymorphic products. Most of the polymorphic fragments contained repetitive sequences. Only one singlecopy sequence fragment was found, a 1.2-kb fragment amplified by primer OPB-19 and subsequently named TGMS1.2. TGMS1.2 was mapped on chromosome 8 with a RIL population and confirmed by remapping with a DHL population. Segregation analysis using TGMS1.2 as a probe indicated that TGMS1.2 both consegregated and was lined with the TGMS gene in this population. It is located about 6.7 cM from the TGMS gene. As TGMS1.2 is linked to the TGMS gene, the TGMS gene must be located on chromosome 8.This research was supported by the Rockefeller Foundation and China National High-Tech Research and Development Program. The first author is a Rockefeller Career Fellow at Texas Tech University     

The Arabidopsis peptide kiss of death is an inducer of programmed cell death

Programmed cell death (PCD) has a key role in defence and development of all multicellular organisms. In plants, there is a large gap in our knowledge of the molecular machinery involved at the various stages of PCD, especially the early steps. Here, we identify kiss of death (KOD) encoding a 25-amino-acid peptide that activates a PCD pathway in Arabidopsis thaliana. Two mutant alleles of KOD exhibited a reduced PCD of the suspensor, a single file of cells that support embryo development, and a reduced PCD of root hairs after a 55°C heat shock. KOD expression was found to be inducible by biotic and abiotic stresses. Furthermore, KOD expression was sufficient to cause death in leaves or seedlings and to activate caspase-like activities. In addition, KOD-induced PCD required light in leaves and was repressed by the PCD-suppressor genes AtBax inhibitor 1 and p35. KOD expression resulted in depolarization of the mitochondrial membrane, placing KOD above mitochondria dysfunction, an early step in plant PCD. A KOD∷GFP fusion, however, localized in the cytosol of cells and not mitochondria.     

Genetic bases of instability of male sterility and fertility reversibility in photoperiod-sensitive genic male-sterile rice

Photoperiod-sensitive genetic male-sterile (PSGMS) rice, with its male fertility regulated by photoperiod length, is very useful for hybrid rice development. However, breeding for new PSGMS lines has faced two major difficulties – the stability of male sterility and the reversibility of male fertility. In this study we assessed the genetic bases of stability of sterility and fertility reversibility using a molecular marker-based approach. A cross was made between two newly bred PSGMS lines: Peiai 64S, which has a stable sterility but is difficult to reverse to fertility, and 8902S, which has a unstable sterility but is easy to reverse to fertility. The fertility of the parents and of the F₁ and F₂ populations was repeatedly examined under 11 different long-day and short-day conditions. The genetic effects were assayed by interval mapping and two-way analyses of variance using the F₂ data of 128 polymorphic loci representing all the 12 rice chromosomes. The analyses resolved a number of single-locus QTLs and two-locus interactions under both long-day and short day conditions. The interactions involved a large number of loci, most of which were not detectable on a single-locus basis. The results showed that the genetic bases of both stability of sterility and reversibility of fertility are the joint effects of the additive effects of the QTLs and additive-by-additive components of two-locus interactions. The implications of these findings in hybrid rice development are also discussed. Received: 11 January 1999 / Accepted: 19 January 1999     

POLLEN STERILITY,a novel suppressor of cell division,is required for timely tapetal programmed cell death in rice

Science China Life Sciences - Timely programmed cell death (PCD) of the tapetum supplying nutrients to microspores is a prerequisite for normal pollen development. Here we identified a unique...     

Transglutaminase activity is involved in polyamine-induced programmed cell death.

Natural polyamines, i.e., putrescine, spermidine, and spermine, are ubiquitous molecules essential for cell proliferation and differentiation. In the present study, the effect of polyamines on primary cultures of bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and a human melanoma cell line was examined. While in the absence of fetal calf serum (FCS) polyamines had no effect on viability, in the presence of FCS spermidine and spermine, at concentrations close to physiologic levels, induced a dose-dependent cell death, whereas putrescine was ineffective. RASMCs were significantly more sensitive than other cells. FACS analysis, oligo-nucleosome ELISA, Hoechst nuclear staining, and Annexin V-FITC quantification showed that cell death was likely due to apoptosis. Cells exposed to spermidine showed a marked increase of intracellular transglutaminase (TGase) activity ( approximately 30-fold over control). Inhibitors of polyamine oxidation or inhibitors of TGase activity prevented polyamine-induced apoptosis. Moreover, tissue TGase overexpression significantly increased cell sensitivity to polyamine, suggesting that this effect is likely related to enhanced intracellular TGase activity. These data indicate that polyamines may modulate cell viability through a novel TGase-dependent process.     

Over-expression of mitochondrial heat shock protein 70 suppresses programmed cell death in rice

In this study, we identified and functionally characterized the mitochondrial heat shock protein 70 (mtHsp70). Over-expression of mtHsp70 suppressed heat- and H₂O₂-induced programmed cell death (PCD) in rice protoplasts, as reflected by higher cell viability, decreased DNA laddering and chromatin condensation. Mitochondrial membrane potential (Δψ_m) after heat shock was destroyed gradually in protoplasts, but mtHsp70 over-expression showed higher Δψ_m relative to the vector control cells, and partially inhibited cytochrome c release from mitochondria to cytosol. Heat treatment also significantly increased reactive oxygen species (ROS) generation, a phenomenon not observed in protoplasts over-expressing mtHsp70. Together, these results suggest that mtHsp70 may suppress PCD in rice protoplasts by maintaining mitochondrial Δψ_m and inhibiting the amplification of ROS.     

Induction of programmed cell death in Arabidopsis and rice by single-wall carbon nanotubes

? Premise of the study: Single-walled carbon nanotubes (SWCNTs) have many unique structural and mechanical properties. Their potential applications, especially in biomedical engineering and medical chemistry, have been increasing in recent years, but the toxicological impact of nanoparticles has rarely been studied in plants. ? Methods: We exposed Arabidopsis and rice leaf protoplasts to SWCNTs and examined cell viability, DNA damage, reactive oxygen species generation, and related gene expression. We also tested the effects of nanoparticles on Arabidopsis leaves after injecting a SWCNT solution. EM-TUNEL (electron-microscopic terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) and a cerium chloride staining method were used. ? Key results: SWCNTs caused adverse cellular responses including cell aggregation, chromatin condensation along with a TUNEL-positive reaction, plasma membrane deposition, and H(2)O(2) accumulation. The effect of SWCNTs on the survival of cells was dose dependent, with 25 μg/mL inducing 25% cell death in 6 h. In contrast, activated carbon, which is not a nano-sized carbon particle, did not induce cell death even 24 h after treatments. The data indicated that the nano-size of the particle is a critical factor for toxicity. Moreover, endocytosis-like structures with cerium chloride deposits formed after SWCNT treatment, suggesting a possible pathway for nanoparticles to traverse the cell membrane. ? Conclusions: Consequently, SWCNTs have an adverse effect on protoplasts and leaves through oxidative stress, leading to a certain amount of programmed cell death. Although nanomaterials have great advantages in many respects, the benefits and side effects still need to be assessed carefully.     

Expression of cysteine proteinase during developmental events associated with programmed cell death in brinjal

Here we show that the expression of a cysteine proteinase coincides with several developmental events associated with programmed cell death (PCD) in Solanum melongena (brinjal), i.e. during leaf senescence, fruit senescence, xylogenesis, nucellar cell degeneration and anther senescence. We have isolated a cDNA encoding brinjal cysteine proteinase (SmCP) that shares high (90-92%) amino acid identity to cysteine proteinases of tobacco (CYP-8) and tomato (LCYP-2) that have not been previously reported to be senescence-associated. In contrast, SmCP shows lower (39-41%) amino acid identity to other senescence-related cysteine proteinases and, unlike most of them, it is not preferentially expressed in certain organs or cell types. Northern analysis of leaves, fruits and flowers at different stages of development showed that SmCP expression increased significantly at senescence in leaf and fruit, but was highly expressed throughout flower development. In situ hybridization studies on flower sections using an antisense RNA probe localized the SmCP mRNA to the xylem, the epidermis and the endothecium of the anther and the nucellar cells, suggesting its involvement in PCD during xylogenesis, anther senescence and ovule development, respectively. Its expression during nucellar cell degeneration suggests that protein reserves of the nucellus are released to the developing embryo. Polarity in its pattern of expression in the nucellus of the developing seed (40DAP) further implies a directional flow of these nutrients.     

Decreased protein kinase C activity is associated with programmed cell death (apoptosis) in freshly isolated rat hepatocytes

Apoptosis of freshly isolated rat hepatocytes was induced by either the omission of fetal bovine serum in the culture medium or addition of the protein kinase C inhibitors polymyxin B or staurosporin. The time-course of DNA breakdown into oligonucleosome-sized fragments and the activity of protein kinase C was determined. Hepatocytes were found to be sensitive to bleomycin which induced a high degree of DNA breakdown even within 30 min incubation. Both staurosporin and polymyxin B induced DNA degradation in hepatocytes after three hours incubation, an effect that was partially prevented by phorbol myristate acetate (PMA). After eight hours incubation, PMA failed to counteract this action and itself produced the apoptosis of rat hepatocytes. The results suggest the involvement of protein kinase C in hepatocyte survival.     

Spatial and temporal progress of programmed cell death in the developing starchy endosperm of rice

Programmed cell death (PCD) is the genetically regulated disassembly of cells, and occurs in the endosperm of cereals during seed maturation. Since PCD determines the lifetime of cells, it can affect endosperm growth and, therefore, cereal yield. However, the features and mechanisms of PCD in the developing starchy endosperm in the Poaceae remain unclear. In the present study, we investigated the characteristics of PCD in developing starchy endosperm of rice (Oryza sativa L.) by fluorescence microscopy, focusing on the spatial and temporal progress of PCD-associated responses. Cell death commenced in the central region of starchy endosperm, and then spread to the peripheral region. PCD-associated responses, such as mitochondrial membrane permeabilization and activation of the protease that cleaves the amino acid sequence VEID, showed similar spatial patterns to that of cell death, but preceded cell death. Degradation of nuclear DNA could not be detected in developing starchy endosperm by the TUNEL assay. These results indicated that PCD in developing starchy endosperm of rice proceeds via a highly organized pattern. In addition, these results suggested that PCD in developing starchy endosperm of rice is characterized by the involvement of mitochondrial signaling and the activity of a caspase-like protease that cleaves the VEID sequence.