Programmed Cell Death In Plants Research Articles

Ethylene-Responsive AP2/ERF Transcription Factor MACD1 Participates in Phytotoxin-Triggered Programmed Cell Death

To investigate plant programmed cell death (PCD), we developed the model system using phytotoxin AAL, which is produced by necrotrophic pathogen Alternaria alternata f. sp. lycopersici, and AAL-sensitive Nicotiana umbratica. We previously reported that ethylene (ET) signaling plays a pivotal role in AAL-triggered cell death (ACD). However, downstream signaling of ET to ACD remains unclear. Here, we show that the modulator of AAL cell death 1 (MACD1), which is an APETALA2/ET response factor (ERF) transcription factor, participates in ACD and acts downstream of ET signaling during ACD. MACD1 is a transcriptional activator and MACD1 overexpression plants showed earlier ACD induction than control plants, suggesting that MACD1 positively regulates factors affecting cell death. To investigate the role of MACD1 in PCD, we used Arabidopsis thaliana and a structural analog of AAL, fumonisin B1 (FB1). FB1-triggered cell death was compromised in ET signaling and erf102 mutants. The loh2 mutants showed sensitivity to AAL, and the loh2-1/erf102 double mutant compromised ACD, indicating that ERF102 also participates in ACD. To investigate the PCD-associated genes regulated by ERF102, we compared our microarray data using ERF102 overexpression plants with the database of upregulated genes by AAL treatment in loh2 mutants, and found genes under the control of ERF102 in ACD.

Aluminum-induced programmed cell death promoted by AhSAG, a senescence-associated gene in Arachis hypoganea L.

Programmed cell death (PCD) is a foundational cellular process in plant development and elimination of damaged cells under environmental stresses. In this study, Al induced PCD in two peanut (Arachis hypoganea L.) cultivars Zhonghua 2 (Al-sensitive) and 99-1507 (Al-tolerant) using DNA ladder, TUNEL detection and electron microscopy. The concentration of Al-induced PCD was lower in Zhonghua 2 than in 99-1507. AhSAG, a senescence-associated gene was isolated from cDNA library of Al-stressed peanut with PCD. Open reading frame (ORF) of AhSAG was 474bp, encoding a SAG protein composed of 157 amino acids. Compared to the control and the antisense transgenic tobacco plants, the fast development and blossom of the sense transgenic plants happened to promote senescence. The ability of Al tolerance in sense transgenic tobacco was lower than in antisense transgenic tobacco according to root elongation and Al content analysis. The expression of AhSAG-GFP was higher in sense transgenic tobacco than in antisense transgenic tobacco. Altogether, these results indicated that there was a negative relationship between Al-induced PCD and Al-resistance in peanut, and the AhSAG could induce or promote the occurrence of PCD in plants

The Plant S1-Like Nuclease Family Has Evolved a Highly Diverse Range of Catalytic Capabilities

Plant S1-like nucleases, often referred to as nuclease I enzymes, are the main class of enzymes involved in nucleic acid degradation during plant programmed cell death. The catalytically active site of these enzymes shows a significant similarity to the well-described P1 nuclease from Penicillium citrinum. Previously published studies reported that plant S1-like nucleases possess catalytic activities similar to their fungal orthologs, i.e. they hydrolyze single-stranded DNA and RNA, and less efficiently double-stranded DNA, in the presence of zinc ions. Here we describe a comprehensive study of the nucleolytic activities of all Arabidopsis S1-like paralogs. Our results revealed that different members of this family are characterized by a surprisingly large variety of catalytic properties. We found that, in addition to Zn(2+)-dependent enzymes, this family also comprises nucleases activated by Ca(2+) and Mn(2+), which implies that the apparently well-known S1 nuclease active site in plant nucleases is able to cooperate with different activatory ions. Moreover, particular members of this class differ in their optimum pH value and substrate specificity. These results shed new light on the widely accepted classification of plant nucleases which is based on the assumption that the catalytic requirements of plant nucleases reflect their phylogenetic origin. Our results imply the need to redefine the understanding of the term 'nuclease I'. Analysis of the phylogenetic relationships between S1-like enzymes shows that plant representatives of this family evolve toward an increase in catalytic diversity. The importance of this process for the biological functions of plant S1-type enzymes is discussed.

Structural insight into plant programmed cell death mediated by BAG proteins inArabidopsis thaliana

The recently identified plant Bcl-2-associated athanogene (BAG) family plays an extensive role in plant programmed cell death (PCD) processes ranging from growth and development to stress responses and even cell death. In the Arabidopsis thaliana BAG (AtBAG) protein family, four members (AtBAG1-4) have a domain organization similar to that of mammalian BAG proteins. Here, crystal structures of the BAG domains (BDs) of AtBAG1-4 have been determined; they have high homology and adopt a structure comprising three short parallel α-helices, similar to some mammalian BAG proteins. The crystal structure of a complex of the AtBAG1 ubiquitin-like domain and BAG domain (UBD) with the Hsc70 nucleotide-binding domain (NBD) was also determined. The binding of the AtBAG1 BD to the Hsc70 NBD induces conformational change of the Hsc70 NBD to the open state and reduces the affinity of the NBD for ADP. In vivo studies showed that bag2-1 mutant plants are larger than wild-type plants when growing under normal conditions, indicating that the AtBAG proteins might regulate plant PCD and confer tolerance to stresses in plants. These structural and functional analyses indicate that the AtBAG proteins function as nucleotide-exchange factors for Hsp70/Hsc70 in A. thaliana and that the mechanism of regulation of chaperone-mediated protein folding is conserved in plants.

Involvement of phospholipase D-related signal transduction in chemical-induced programmed cell death in tomato cell cultures

Phospholipase D (PLD) and its product phosphatidic acid (PA) are incorporated in a complex metabolic network in which the individual PLD isoforms are suggested to regulate specific developmental and stress responses, including plant programmed cell death (PCD). Despite the accumulating knowledge, the mechanisms through which PLD/PA operate during PCD are still poorly understood. In this work, the role of PLDα1 in PCD and the associated caspase-like proteolysis, ethylene and hydrogen peroxide (H(2)O(2)) synthesis in tomato suspension cells was studied. Wild-type (WT) and PLDα1-silenced cell lines were exposed to the cell death-inducing chemicals camptothecin (CPT), fumonisin B1 (FB1) and CdSO(4). A range of caspase inhibitors effectively suppressed CPT-induced PCD in WT cells, but failed to alleviate cell death in PLDα1-deficient cells. Compared to WT, in CPT-treated PLDα1 mutant cells, reduced cell death and decreased production of H(2)O(2) were observed. Application of ethylene significantly enhanced CPT-induced cell death both in WT and PLDα1 mutants. Treatments with the PA derivative lyso-phosphatidic acid and mastoparan (agonist of PLD/PLC signalling downstream of G proteins) caused severe cell death. Inhibitors, specific to PLD and PLC, remarkably decreased the chemical-induced cell death. Taken together with our previous findings, the results suggest that PLDα1 contributes to caspase-like-dependent cell death possibly communicated through PA, reactive oxygen species and ethylene. The dead cells expressed morphological features of PCD such as protoplast shrinkage and nucleus compaction. The presented findings reveal novel elements of PLD/PA-mediated cell death response and suggest that PLDα1 is an important factor in chemical-induced PCD signal transduction.

Pattern-Triggered Immunity Suppresses Programmed Cell Death Triggered by Fumonisin B1

Programmed cell death (PCD) is a crucial process for plant innate immunity and development. In plant innate immunity, PCD is believed to prevent the spread of pathogens from the infection site. Although proper control of PCD is important for plant fitness, we have limited understanding of the molecular mechanisms regulating plant PCD. Plant innate immunity triggered by recognition of effectors (effector-triggered immunity, ETI) is often associated with PCD. However pattern-triggered immunity (PTI), which is triggered by recognition of elicitors called microbe-associated molecular patterns (MAMPs), is not. Therefore we hypothesized that PTI might suppress PCD. Here we report that PCD triggered by the mycotoxin fumonisin B1 (FB1) can be suppressed by PTI in Arabidopsis. FB1-triggered cell death was suppressed by treatment with the MAMPs flg22 (a part of bacterial flagellin) or elf18 (a part of the bacterial elongation factor EF-Tu) but not chitin (a component of fungal cell walls). Although plant hormone signaling is associated with PCD and PTI, both FB1-triggered cell death and suppression of cell death by flg22 treatment were still observed in mutants deficient in jasmonic acid (JA), ethylene (ET) and salicylic acid (SA) signaling. The MAP kinases MPK3 and MPK6 are transiently activated and inactivated within one hour during PTI. We found that FB1 activated MPK3 and MPK6 about 36–48 hours after treatment. Interestingly, this late activation was attenuated by flg22 treatment. These results suggest that PTI suppression of FB1-triggered cell death may involve suppression of MPK3/MPK6 signaling but does not require JA/ET/SA signaling.

UDP-Glucose Pyrophosphorylase Is a Novel Plant Cell Death Regulator

Programmed cell death (PCD) is an essential process that functions in plant organ sculpture, tissue differentiation, nutrient recycling, and defense against pathogen attack. A full understanding of the mechanism of PCD in plants is hindered by the limited identification of protein components of the complex signaling circuitry that underpins this important physiological process. Here we have used Arabidopsis thaliana and fumonisin B1 (FB1) to identify proteins that constitute part of the PCD signaling network. We made an inadvertent, but important observation that exogenous sucrose modulates FB1-induced cell death and identified sucrose-induced genes from publicly available transcriptomic data sets for reverse genetic analyses. Using transfer-DNA gene knockout plants, UDP-glucose pyrophosphorylase 1 (UGP1), a sucrose-induced gene, was demonstrated to be a critical factor that regulates FB1-induced PCD. We employed 2D-DiGE to identify proteomic changes preceding PCD after exposure of Arabidopsis to FB1 and used UGP1 knockout plants to refine the analysis and isolate downstream candidate proteins with a putative PCD regulatory function. Our results reveal chloroplasts as the predominantly essential organelles in FB1-induced PCD. Overall, this study reveals a novel function of UGP1 as a cell death regulator and provides candidate proteins likely recruited downstream in the activation of plant PCD.

Unveiling Interactions among Mitochondria, Caspase-Like Proteases, and the Actin Cytoskeleton during Plant Programmed Cell Death (PCD)

Aponogeton madagascariensis produces perforations over its leaf surface via programmed cell death (PCD). PCD begins between longitudinal and transverse veins at the center of spaces regarded as areoles, and continues outward, stopping several cells from these veins. The gradient of PCD that exists within a single areole of leaves in an early stage of development was used as a model to investigate cellular dynamics during PCD. Mitochondria have interactions with a family of proteases known as caspases, and the actin cytoskeleton during metazoan PCD; less is known regarding these interactions during plant PCD. This study employed the actin stain Alexa Fluor 488 phalloidin, the actin depolymerizer Latrunculin B (Lat B), a synthetic caspase peptide substrate and corresponding specific inhibitors, as well as the mitochondrial pore inhibitor cyclosporine A (CsA) to analyze the role of these cellular constituents during PCD. Results depicted that YVADase (caspase-1) activity is higher during the very early stages of perforation formation, followed by the bundling and subsequent breakdown of actin. Actin depolymerization using Lat B caused no change in YVADase activity. In vivo inhibition of YVADase activity prevented PCD and actin breakdown, therefore substantiating actin as a likely substrate for caspase-like proteases (CLPs). The mitochondrial pore inhibitor CsA significantly decreased YVADase activity, and prevented both PCD and actin breakdown; therefore suggesting the mitochondria as a possible trigger for CLPs during PCD in the lace plant. To our knowledge, this is the first in vivo study using either caspase-1 inhibitor (Ac-YVAD-CMK) or CsA, following which the actin cytoskeleton was examined. Overall, our findings suggest the mitochondria as a possible upstream activator of YVADase activity and implicate these proteases as potential initiators of actin breakdown during perforation formation via PCD in the lace plant.

TGBp3 triggers the unfolded protein response and SKP1‐dependent programmed cell death

The Potato virus X (PVX) triple gene block protein 3 (TGBp3), an 8-kDa membrane binding protein, aids virus movement and induces the unfolded protein response (UPR) during PVX infection. TGBp3 was expressed from the Tobacco mosaic virus (TMV) genome (TMV-p3), and we noted the up-regulation of SKP1 and several endoplasmic reticulum (ER)-resident chaperones, including the ER luminal binding protein (BiP), protein disulphide isomerase (PDI), calreticulin (CRT) and calmodulin (CAM). Local lesions were seen on leaves inoculated with TMV-p3, but not TMV or PVX. Such lesions were the result of TGBp3-elicited programmed cell death (PCD), as shown by an increase in reactive oxygen species, DNA fragmentation and induction of SKP1 expression. UPR-related gene expression occurred within 8 h of TMV-p3 inoculation and declined before the onset of PCD. TGBp3-mediated cell death was suppressed in plants that overexpressed BiP, indicating that UPR induction by TGBp3 is a pro-survival mechanism. Anti-apoptotic genes Bcl-xl, CED-9 and Op-IAP were expressed in transgenic plants and suppressed N gene-mediated resistance to TMV, but failed to alleviate TGBp3-induced PCD. However, TGBp3-mediated cell death was reduced in SKP1-silenced Nicotiana benthamiana plants. The combined data suggest that TGBp3 triggers the UPR and elicits PCD in plants.

EAT1 promotes tapetal cell death by regulating aspartic proteases during male reproductive development in rice

Programmed cell death is essential for the development of multicellular organisms, yet pathways of plant programmed cell death and its regulation remain elusive. Here we report that ETERNAL TAPETUM 1, a basic helix-loop-helix transcription factor conserved in land plants, positively regulates programmed cell death in tapetal cells in rice anthers. eat1 exhibits delayed tapetal cell death and aborted pollen formation. ETERNAL TAPETUM 1 directly regulates the expression of OsAP25 and OsAP37, which encode aspartic proteases that induce programmed cell death in both yeast and plants. Expression and genetic analyses revealed that ETERNAL TAPETUM 1 acts downstream of TAPETUM DEGENERATION RETARDATION, another positive regulator of tapetal programmed cell death, and that ETERNAL TAPETUM 1 can also interact with the TAPETUM DEGENERATION RETARDATION protein. This study demonstrates that ETERNAL TAPETUM 1 promotes aspartic proteases triggering plant programmed cell death, and reveals a dynamic regulatory cascade in male reproductive development in rice.

Mitochondrial VDAC and hexokinase together modulate plant programmed cell death

The voltage-dependent anion channel (VDAC) and mitochondrially located hexokinase have been implicated both in pathways leading to cell death on the one hand, and immortalization in tumor formation on the other. While both proteins have also been implicated in death processes in plants, their interaction has not been explored. We have examined cell death following heterologous expression of a rice VDAC in the tobacco cell line BY2 and in leaves of tobacco plants and show that it is ameliorated by co-expression of hexokinase. Hexokinase also abrogates death induced by H2O2. We conclude that the ratio of expression of the two proteins and their interaction play a major role in modulating death pathways in plants.

Programmed cell death in plants

The modern concepts of programmed cell death (PCD) in plants are reviewed as compared to PCD (apoptosis) in animals. Special attention is focused on considering the potential mechanisms of implementation of this fundamental biological process and its participants. In particular, the proteolytic enzymes involved in PCD in animals (caspases) and plants (phytaspases) are compared. Emphasis is put on elucidation of both common features and substantial differences of PCD implementation in plants and animals.

The involvement of ethylene in programmed cell death and climacteric-like behaviour during the remodelling of lace plant (Aponogeton madagascariensis) leaves

Programmed cell death (PCD) plays an important role in several plant developmental processes. The phytohormone ethylene has been implicated in PCD signalling in many plant systems, but it is also important in developmental processes such as seed germination, flowering, and climacteric fruit ripening. Lace plant (Aponogeton madagascariensis (Mirbel) H. Bruggen) is an aquatic monocot that develops perforated leaves via the deletion of cells through developmentally regulated PCD. The plant is ideal for studying PCD; however, little is known about the regulation of cellular death involved in this system. The current study examines ethylene as a potential signalling molecule in lace plant PCD and investigates climacteric-like behaviour during lace plant leaf development. Whole plants were treated with the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG), the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), or a combination of both. Subsequently, ethylene levels were monitored, and leaf development was analyzed. The results indicate that ethylene is involved in lace plant PCD signalling. AVG-treated plants had significantly lower ethylene outputs and a significant reduction in perforation formation. The inhibitory effect of AVG was recovered when AVG and ACC were applied simultaneously. The data presented here show for the first time, to our knowledge, climacteric-like behaviour during the remodelling of leaves.

DEVDase activity is induced in potato leaves during Phytophthora infestans infection

Programmed cell death (PCD) occurs in plants, animals and several branches of unicellular eukaryotes as a part of developmental and/or defense processes. Caspase proteases are universal mediators of animal apoptosis, a type of PCD. In plants, there are not animal caspase homologs; therefore, the characterization of caspase-like activities is of considerable importance to our understanding of PCD in plants. Here we report for the first time the involvement of caspase-3-like activity in the resistance mechanism of potato to Phytophthora infestans infection. We showed that disease development in infected potato leaves is dependent of caspase-3-like activity. Unlike plant DEVDases previously reported, this DEVDase activity was sensitive to the serine protease inhibitor PMSF. As reported for other subtilisin- like proteases with caspase activity, potato DEVDase activity was mainly localized in the apoplast. We demonstrated that in total protein extract DEVDase activity accounts for a 60% of serine proteases; however, this percentage increases to 100% in the apoplast. Additionally, this caspase-3-like activity is constitutively expressed in the apoplast of potato leaves. Total DEVDase activity is induced only in potato cultivars with high field resistance to P. infestans. These results show that potato caspase-3-like protease could constitute a tool in the potato defense mechanisms resulting in partial resistance, although further assays would be necessary in order to elucidate its role.

Molecular cloning of the apoptosis-related calcium-binding protein AsALG-2 in Avena sativa.

Victorin, the host-selective toxin produced by the fungus Cochliobolus victoriae, induces programmed cell death (PCD) in victorin-sensitive oat lines with characteristic features of animal apoptosis, such as mitochondrial permeability transition, chromatin condensation, nuclear DNA laddering and rRNA/mRNA degradation. In this study, we characterized a calcium-binding protein, namely AsALG-2, which might have a role in the victorin-induced PCD. AsALG-2 is homologous to the Apoptosis-Linked Gene ALG-2 identified in mammalian cells. Northern blot analysis revealed that the accumulation of AsALG-2 transcripts increased during victorin-induced PCD, but not during necrotic cell death. Salicylic acid, chitosan and chitin strongly activated the expression of general defence response genes, such as PR-10; however, neither induced cell death nor the accumulation of AsALG-2 mRNA. Pharmacological studies indicated that victorin-induced DNA laddering and AsALG-2 expression were regulated through similar pathways. The calcium channel blocker, nifedipine, moderately inhibited the accumulation of AsALG-2 mRNA during cell death. Trifluoperazine (calmodulin antagonist) and K252a (serine-threonine kinase inhibitor) reduced the victorin-induced phytoalexin accumulation, but did not prevent the victorin-induced DNA laddering or accumulation of AsALG-2 mRNA. Taken together, our investigations suggest that there is a calcium-mediated signalling pathway in animal and plant PCD in common.

Programmed cell death in plants: lessons from bacteria?

Programmed cell death (PCD) has well-established roles in the development and physiology of animals, plants, and fungi. Although aspects of PCD control appear evolutionarily conserved between these organisms, the extent of conservation remains controversial. Recently, a putative bacterial PCD protein homolog in plants was found to play a significant role in cell death control, indicating a conservation of function between these highly divergent organisms. Interestingly, these bacterial proteins are thought to be evolutionarily linked to the Bcl-2 family of proteins. In this opinion article, we propose a new unifying model to describe the relationship between bacterial and plant PCD systems and propose that the underlying control of PCD is conserved across at least three Kingdoms of life.

Cloning and characterization of two rice long-chain base kinase genes and their function in disease resistance and cell death

Sphingolipid metabolites such as long-chain base 1-phosphates (LCBPs) have been shown to play an important role in plants; however, little is known about their function in plant disease resistance and programmed cell death (PCD). In the present study, we cloned and identified two rice long-chain base kinase (LCBK) genes (OsLCBK1 and OsLCBK2), which are involved in biosynthesis of LCBPs, and performed functional analysis in transgenic tobacco. Expression of OsLCBK1 and OsLCBK2 was induced in rice seedlings after treatments with defense signaling molecules and after infection by Magnaporthe grisea, the causal agent of blast disease. Transgenic tobacco plants overexpressing OsLCBK1 were generated and disease resistance assays indicate that the OsLCBK1-overexpressing plants showed enhanced disease resistance against Pseudmonas syringae pv. tabacci, the causal agent of wildfire disease, and tobacco mosaic virus. Expression levels of some defense-related genes were constitutively up-regulated and further induced after pathogen infection in the OsLCBK1-overexpressing plants. Treatment with fungal toxin fumonisin B1, an effective inducer of PCD in plants, resulted in reduced level of cell death in the OsLCBK1-overexpressing plants, as indicated by cell death staining, leakage of electrolyte and expression of hypersensitive response indicator genes. These data suggest that rice LCBKs, probably through regulation of endogenous LCBP level, play important roles in disease resistance response and PCD in plants.

Plant Programmed Cell Death Caused by an Autoactive Form of Prf Is Suppressed by Co-Expression of the Prf LRR Domain

In tomato, the NBARC-LRR resistance (R) protein Prf acts in concert with the Pto or Fen kinase to determine immunity against Pseudomonas syringae pv. tomato (Pst). Prf-mediated defense signaling is initiated by the recognition of two sequence-unrelated Pst-secreted effector proteins, AvrPto and AvrPtoB, by tomato Pto or Fen. Prf detects these interactions and activates signaling leading to host defense responses including localized programmed cell death (PCD) that is associated with the arrest of Pst growth. We found that Prf variants with single amino acid substitutions at D1416 in the IHD motif (isoleucine-histidine-aspartic acid) in the NBARC domain cause effector-independent PCD when transiently expressed in leaves of Nicotiana benthamiana, suggesting D1416 plays an important role in activation of Prf. The N-terminal region of Prf (NPrf) and the LRR domain are required for this autoactive Prf cell death signaling but dispensable for accumulation of the Prf(D1416V) protein. Significantly, co-expression of the Prf LRR but not NPrf, with Prf(D1416V), AvrPto/Pto, AvrPtoB/Pto, an autoactive form of Pto (Pto(Y207D)), or Fen completely suppresses PCD. However, the Prf LRR does not interfere with PCD caused by Rpi-blb1(D475V), a distinct R protein-mediated PCD signaling event, or that caused by overexpression of MAPKKKα, a protein acting downstream of Prf. Furthermore, we found the Prf(D1416V) protein is unable to accumulate in plant cells when co-expressed with the Prf LRR domain, likely explaining the cell death suppression. The mechanism for the LRR-induced degradation of Prf(D1416V) is unknown but may involve interference in the intramolecular interactions of Prf or to binding of the unattached LRR to other host proteins that are needed for Prf stability.

14-3-3-Regulated Ca2+-dependent protein kinase CPK3 is required for sphingolipid-induced cell death in Arabidopsis

In eukaryotic cells, sphingoid long chain bases (LCBs) such as sphingosine or phytosphingosine (PHS) behave as second messengers involved in various processes including programmed cell death (PCD). In plants, induction of PCD by LCBs has now been described, but the signalling pathway is still enigmatic. Using Arabidopsis, we identify new key steps in this pathway. We demonstrate that PHS induces activation of the calcium-dependent kinase CPK3, which phosphorylates its binding partners, the 14-3-3 proteins. This phosphorylation leads to the disruption of the complex and to CPK3 degradation. Using cpk3 knockout lines, we demonstrate that CPK3 is a positive regulator of LCB-mediated PCD. These findings establish 14-3-3-regulated CPK3 as a key component of the LCB pathway leading to PCD in plants.

Sorting out the role of nitric oxide in cadmium-induced Arabidopsis thaliana programmed cell death

As a vital cell-signaling molecule, nitric oxide (NO) has been reported to regulate toxic metal responses in plants. Our recent report has suggested that caspase-3-like protease activation was detected in Arabidopsis (Arabidopsis thaliana) after Cd2+ treatment. NO contributed caspase-3-like protease activation in Cd2+ induced Arabidopsis thaliana programmed cell death (PCD), which was mediated by MPK6. It was first shown that NO promotes Cd2+-induced Arabidopsis PCD by promoting MPK6-mediated caspase-3-like activation. Our study contributed to the understanding of NO signaling pathway in Cd2+-induced Arabidopsis thaliana PCD. Although several studies have revealed that NO regulates plant PCD, compared with the study of signaling pathways involved in animal cell apoptosis, the mechanism of NO function still remains elusive and the molecular mechanisms of MAPK are far from clear in Cd2+-induced PCD. By using the fluorescence techniques and the Arabidopsis seedlings as the reference model, the subsequent researches have been performed to obtain comprehensive understanding of Cd2+-induced plant PCD.