Dysfunction of Arabidopsis MACPF domain protein activates programmed cell death via tryptophan metabolism in MAMP‐triggered immunity

Plant immune responses triggered upon recognition of microbe‐associated molecular patterns (MAMPs) typically restrict pathogen growth without a host cell death response. We isolated two Arabidopsis mutants, derived from accession Col‐0, that activated cell death upon inoculation with nonadapted fungal pathogens. Notably, the mutants triggered cell death also when treated with bacterial MAMPs such as flg22. Positional cloning identified NSL1 (Necrotic Spotted Lesion 1) as a responsible gene for the phenotype of the two mutants, whereas nsl1 mutations of the accession No‐0 resulted in necrotic lesion formation without pathogen inoculation. NSL1 encodes a protein of unknown function containing a putative membrane‐attack complex/perforin (MACPF) domain. The application of flg22 increased salicylic acid (SA) accumulation in the nsl1 plants derived from Col‐0, while depletion of isochorismate synthase 1 repressed flg22‐inducible lesion formation, indicating that elevated SA is needed for the cell death response. nsl1 plants of Col‐0 responded to flg22 treatment with an RBOHD‐dependent oxidative burst, but this response was dispensable for the nsl1‐dependent cell death. Surprisingly, loss‐of‐function mutations in PEN2, involved in the metabolism of tryptophan (Trp)‐derived indole glucosinolates, suppressed the flg22‐induced and nsl1‐dependent cell death. Moreover, the increased accumulation of SA in the nsl1 plants was abrogated by blocking Trp‐derived secondary metabolite biosynthesis, whereas the nsl1‐dependent hyperaccumulation of PEN2‐dependent compounds was unaffected when the SA biosynthesis pathway was blocked. Collectively, these findings suggest that MAMP‐triggered immunity activates a genetically programmed cell death in the absence of the functional MACPF domain protein NSL1 via Trp‐derived secondary metabolite‐mediated activation of the SA pathway.     

Cotton GhCKI disrupts normal male reproduction by delaying tapetum programmed cell death via inactivating starch synthase

Anther infertility under high temperature (HT) conditions is a critical factor contributing to yield loss in cotton (Gossypium hirsutum). Using large‐scale expression profile sequencing, we studied the effect of HT on cotton anther development. Our analysis revealed that altered carbohydrate metabolism or disrupted tapetal programmed cell death (PCD) underlie anther sterility. Expression of the Gossypium hirsutum casein kinase I (GhCKI) gene, which encodes a homolog of casein kinase I (CKI), was induced in an HT‐sensitive cotton line after exposure to HT. As mammalian homologs of GhCKI are involved in inactivation of glycogen synthase and the regulation of apoptosis, GhCKI may be considered a target gene for improving anther fertility under HT conditions. Our studies suggest that GhCKI exhibits starch synthase kinase activity, increases glucose content in early‐stage buds and activates the accumulation of abscisic acid, thereby disturbing the balance of reactive oxygen species and eventually disrupting tapetal PCD, leading to anther abortion or indehiscence. These results indicate that GhCKI may be a key regulator of tapetal PCD and anther dehiscence, with the potential to facilitate regulation of HT tolerance in crops.     

Dynamic infection of Verticillium dahliae in upland cotton

• Verticillium wilt, an infection caused by the soilborne fungus Verticillium dahliae, is one of the most serious diseases in cotton. No effective control method against V. dahliae has been established, and the infection mechanism of V. dahliae in upland cotton remains unknown.
• GFP‐tagged V. dahliae isolates with different pathogenic abilities were used to analyse the colonisation and infection of V. dahliae in the roots and leaves of different upland cotton cultivars, the relationships among infection processes, the immune responses and the resistance ability of different cultivars against V. dahliae.
• Here, we report a new infection model for V. dahliae in upland cotton plants. V. dahliae can colonise and infect any organ of upland cotton plants and then spread to the entire plant from the infected organ through the surface and interior of the organ.
• Vascular tissue was found to not be the sole transmission route of V. dahliae in cotton plants. In addition, the rate of infection of a V. dahliae isolate with strong pathogenicity was notably faster than that of an isolate with weak pathogenicity. The resistance of upland cotton to Verticillium wilt was related to the degree of the immune response induced in plants infected with V. dahliae. These results provide a theoretical basis for studying the mechanism underlying the interaction between V. dahliae and upland cotton. These results provide a theoretical basis for studying the mechanism underlying the interaction between V. dahliae and upland cotton.

     

Interplay of cell proliferation and cell death in Drosophila tissue regeneration

Regeneration is a fascinating process that allows some organisms to reconstruct damaged tissues. In addition to the classical regeneration model of the Drosophila larval imaginal discs, the genetically induced tissue ablation model has promoted the understanding of molecular mechanisms underlying cell death, proliferation, and remodeling for tissue regeneration. Recent studies have also revealed that tissue injury responses occur not only locally but also systemically, even in the uninjured region. Genetic studies in Drosophila have demonstrated the dynamic role of the cell death‐induced tissue response in the reconstruction of damaged tissues.     

Temporal rhythm of petal programmed cell death in Ipomoea purpurea

• Flowers are the main sexual reproductive organs in plants. The shapes, colours and scents of corolla of plant flowers are involved in attracting insect pollinators and increasing reproductive success. The process of corolla senescence was investigated in Ipomoea purpurea (Convolvulaceae) in this study.
• In the research methods of plant anatomy, cytology, cell chemistry and molecular biology were used.
• The results showed that at the flowering stage cells already began to show distortion, chromatin condensation, mitochondrial membrane degradation and tonoplast dissolution and rupture. At this stage genomic DNA underwent massive but gradual random degradation. However, judging from the shape and structure, aging characteristics did not appear until the early flower senescence stage. The senescence process was slow, and it was completed at the late stage of flower senescence with a withering corolla.
• We may safely arrive at the conclusion that corolla senescence of I. purpurea was mediated by programmed cell death (PCD) that occurred at the flowering stage. The corolla senescence exhibited an obvious temporal rhythm, which demonstrated a high degree of coordination with pollination and fertilization.

     

Effect of precursor feeding on alkaloid accumulation by a strictosidine synthase over-expressing transgenic cell line S1 of Catharanthus roseus

The transgenic S1 cell line of Catharanthus roseus (L.) G. Don has been used to study possible rate limiting steps in the terpenoid indole alkaloid (TIA) biosynthesis. Line S1 carries a recombinant, over-expressed version of the endogenous Str gene which encodes strictosidine synthase (STR; EC 4.3.3.2). STR catalyzes the stereospecific condensation of tryptamine and secologanin to strictosidine. Various concentrations and combinations of biosynthetic indole precursors L-tryptophan, tryptamine, and iridoid precursors loganin and secologanin were added to the cell suspension cultures of line S1. The largest TIA accumulation occurred when the precursor was supplied at the time of inoculation of the cells into the production medium. Line S1 could supply tryptamine endogenously up to 0.8 mM loganin feeding. The enhancement of the accumulation of TIAs by addition of loganin indicates a limitation in the terpenoid pathway. Supplying tryptamine or tryptophan along with the iridoid precursors resulted in even further increase of alkaloid accumulation. Under optimal conditions, cultures of line S1 accumulated about 600 mol l^–1 of TIAs. Also, the conversion of strictosidine into other TIAs further down the pathway seems to be a limiting step. Considering the mass balance of the intermediates fed and TIAs recovered, several yet unknown pathways must be involved in channeling away intermediates from the TIA pathway and in the breakdown of the TIAs. Our results suggest that high rates of tryptamine synthesis can still take place under conditions of low TDC activity and the flux towards tryptamine is induced by loganin feeding. However, accumulation of tryptamine seems to reduce the flux through feedback inhibition.     

Role of the penetration‐resistance genes PEN1, PEN2 and PEN3 in the hypersensitive response and race‐specific resistance in Arabidopsis thaliana

Plants are highly capable of recognizing and defending themselves against invading microbes. Adapted plant pathogens secrete effector molecules to suppress the host's immune system. These molecules may be recognized by host‐encoded resistance proteins, which then trigger defense in the form of the hypersensitive response (HR) leading to programmed cell death of the host tissue at the infection site. The three proteins PEN1, PEN2 and PEN3 have been found to act as central components in cell wall‐based defense against the non‐adapted powdery mildew Blumeria graminis fsp. hordei (Bgh). We found that loss of function mutations in any of the three PEN genes cause decreased hypersensitive cell death triggered by recognition of effectors from oomycete and bacterial pathogens in Arabidopsis. There were considerable additive effects of the mutations. The HR induced by recognition of AvrRpm1 was almost completely abolished in the pen2 pen3 and pen1 pen3 double mutants and the loss of cell death could be linked to indole glucosinolate breakdown products. However, the loss of the HR in pen double mutants did not affect the plants' ability to restrict bacterial growth, whereas resistance to avirulent isolates of the oomycete Hyaloperonospora arabidopsidis was strongly compromised. In contrast, the double and triple mutants demonstrated varying degrees of run‐away cell death in response to Bgh. Taken together, our results indicate that the three genes PEN1, PEN2 and PEN3 extend in functionality beyond their previously recognized functions in cell wall‐based defense against non‐host pathogens.     

Disruption of gene SPL35, encoding a novel CUE domain‐containing protein,leads to cell death and enhanced disease response in rice

Lesion mimic mutants that exhibit spontaneous hypersensitive response (HR)‐like necrotic lesions are ideal experimental systems for elucidating molecular mechanisms involved in plant cell death and defence responses. Here we report identification of a rice lesion mimic mutant, spotted leaf 35 (spl35), and cloning of the causal gene by TAIL‐PCR strategy. spl35 exhibited decreased chlorophyll content, higher accumulation of H₂O₂, up‐regulated expression of defence‐related marker genes, and enhanced resistance to both fungal and bacterial pathogens of rice. The SPL35 gene encodes a novel CUE (coupling of ubiquitin conjugation to ER degradation) domain‐containing protein that is predominantly localized in cytosol, ER and unknown punctate compartment(s). SPL35 is constitutively expressed in all organs, and both overexpression and knockdown of SPL35 cause the lesion mimic phenotype. SPL35 directly interacts with the E2 protein OsUBC5a and the coatomer subunit delta proteins Delta‐COP1 and Delta‐COP2 through the CUE domain, and down‐regulation of these interacting proteins also cause development of HR‐like lesions resembling those in spl35 and activation of defence responses, indicating that SPL35 may be involved in the ubiquitination and vesicular trafficking pathways. Our findings provide insight into a role of SPL35 in regulating cell death and defence response in plants.     

Loss of fumarylacetoacetate hydrolase causes light‐dependent increases in protochlorophyllide and cell death in Arabidopsis

Fumarylacetoacetate hydrolase (FAH) catalyses the final step of the tyrosine degradation pathway, which is essential to animals but was of unknown importance in plants until we found that mutation of Short‐day Sensitive Cell Death1 (SSCD1), encoding Arabidopsis FAH, results in cell death under short‐day conditions. The sscd1 mutant accumulates succinylacetone (SUAC), an abnormal metabolite caused by loss of FAH. Succinylacetone is an inhibitor of δ‐aminolevulinic acid (ALA) dehydratase (ALAD), which is involved in chlorophyll (Chl) biosynthesis. In this study, we investigated whether sscd1 cell death is mediated by Chl biosynthesis and found that ALAD activity is repressed in sscd1 and that protochlorophyllide (Pchlide), an intermediate of Chl biosynthesis, accumulates at lower levels in etiolated sscd1 seedlings. However, it was interesting that Pchlide in sscd1 might increase after transfer from light to dark and that HEMA1 and CHLH are upregulated in the light–dark transition before Pchlide levels increased. Upon re‐illumination after Pchlide levels had increased, reactive oxygen species marker genes, including singlet oxygen‐induced genes, are upregulated, and the sscd1 cell death phenotype appears. In addition, Arabidopsis WT seedlings treated with SUAC mimic sscd1 in decline of ALAD activity and accumulation of Pchlide as well as cell death. These results demonstrate that increase in Pchlide causes cell death in sscd1 upon re‐illumination and suggest that a decline in the Pchlide pool due to inhibition of ALAD activity by SUAC impairs the repression of ALA synthesis from the light–dark transition by feedback control, resulting in activation of the Chl biosynthesis pathway and accumulation of Pchlide in the dark.     

Long-term instability of alkaloid production by stably transformed cell lines of Catharanthus roseus

The productivity of several transgenic cell lines of Catharanthus roseus was monitored over a period of 30 months. The transgenic cultures were obtained by Agrobacterium-mediated transformation of leaf explants with constructs containing recombinant versions of the endogenous Str and Tdc genes, which, respectively, encode strictosidine synthase (STR) and tryptophan decarboxylase (TDC). The expression of these transgenes and the -glucuronidase marker gene were also measured periodically, at the enzymatic level, during this time. Cultures were maintained in selective medium containing either hygromycin or kanamycin and showed GUS activity in the presence of X-gluc, indicating that they carried functional transgenes. The activities of STR and TDC varied greatly over time, occasionally falling to levels not significantly different from those of non-transgenic cultures, and showed susceptibility to the composition of the culture medium. Despite maintaining their transgenic character, the cell lines gradually lost the ability to accumulate terpenoid indole alkaloids (TIAs). The diversity of alkaloids produced was also negatively affected by long-term subculture. We conclude that a strategy of indirect selection, such as the use of antibiotic-resistance genes, is insufficient to maintain the concerted expression of TIA-pathway elements necessary for high productivity.     

Phospholipid:diacylglycerol acyltransferase‐mediated triacylglycerol biosynthesis is crucial for protection against fatty acid‐induced cell death in growing tissues of Arabidopsis

Phospholipid:diacylglycerol acyltransferase (PDAT) and diacylglycerol:acyl CoA acyltransferase play overlapping roles in triacylglycerol (TAG) assembly in Arabidopsis, and are essential for seed and pollen development, but the functional importance of PDAT in vegetative tissues remains largely unknown. Taking advantage of the Arabidopsis tgd1–1 mutant that accumulates oil in vegetative tissues, we demonstrate here that PDAT1 is crucial for TAG biosynthesis in growing tissues. We show that disruption of PDAT1 in the tgd1–1 mutant background causes serious growth retardation, gametophytic defects and premature cell death in developing leaves. Lipid analysis data indicated that knockout of PDAT1 results in increases in the levels of free fatty acids (FFAs) and diacylglycerol. In vivo ¹⁴C‐acetate labeling experiments showed that, compared with wild‐type, tgd1–1 exhibits a 3.8‐fold higher rate of fatty acid synthesis (FAS), which is unaffected by disruption or over‐expression of PDAT1, indicating a lack of feedback regulation of FAS in tgd1–1. We also show that detached leaves of both pdat1–2 and tgd1–1 pdat1–2 display increased sensitivity to FFA but not to diacylglycerol. Taken together, our results reveal a critical role for PDAT1 in mediating TAG synthesis and thereby protecting against FFA‐induced cell death in fast‐growing tissues of plants.     

Oocyte aging in a marine protostome worm: The roles of maturation‐promoting factor and extracellular signal regulated kinase form of mitogen‐activated protein kinase

The roles of maturation‐promoting factor (MPF) and an extracellular signal regulated kinase form of mitogen‐activated protein kinase (ERK MAPK) are analyzed during oocyte aging in the marine protostome worm Cerebratulus. About a day after removal from the ovary, unfertilized metaphase‐I‐arrested oocytes of Cerebratulus begin to flatten and swell before eventually lysing, thereby exhibiting characteristics of a necroptotic mode of regulated cell death. Based on immunoblots probed with phospho‐specific antibodies, MPF and ERK are initially active in freshly mature specimens. However, as oocytes age, both kinase activities decline, with ERK deactivation occurring well before MPF downregulation. Experiments using pharmacological modulators indicate that oocyte degradation is promoted by the maturation‐initiated activation of ERK as well as by the deactivation of MPF that occurs in extensively aged specimens. The potential significance of these findings is discussed relative to previously published results for apoptotic eggs and oocytes of echinoderm and vertebrate deuterostomes.     

Overexpression of FBR41 enhances resistance to sphinganine analog mycotoxin‐induced cell death and Alternaria stem canker in tomato

Fumonisin B1 (FB1) and Alternaria alternate f. sp. lycopersici (AAL)‐toxin are classified as sphinganine analog mycotoxins (SAMTs), which induce programmed cell death (PCD) in plants and pose health threat to humans who consume the contaminated crop products. Herein, Fumonisin B1 Resistant41 (FBR41), a dominant mutant allele, was identified by map‐based cloning of Arabidopsis FB1‐resistant mutant fbr41, then ectopically expressed in AAL‐toxin sensitive tomato (Solanum lycopersicum) cultivar. FBR41‐overexpressing tomato plants exhibited less severe cell death phenotype upon AAL‐toxin treatment. Analysis of free sphingoid bases showed that both fbr41 and FBR41‐overexpressing tomato plants accumulated less sphinganine and phytosphingosine upon FB1 and AAL‐toxin treatment, respectively. Alternaria stem canker is a disease caused by AAL and responsible for severe economic losses in tomato production, and FBR41‐overexpressing tomato plants exhibited enhanced resistance to AAL with decreased fungal biomass and less cell death, which was accompanied by attenuated accumulation of free sphingoid bases and jasmonate (JA). Taken together, our results indicate that FBR41 is potential in inhibiting SAMT‐induced PCD and controlling Alternaria stem canker in tomato.     

Mechanism of cell death and disease resistance induction by transgenic expression of bacterio-opsin

One of the earliest signal transduction events that trigger the hypersensitive response (HR) of plants against pathogen attack is thought to be an alteration of proton flux across the plasma membrane (PM). However, no direct genetic evidence for the involvement of PM-localised proton channels or pumps in the induction of this response has been reported. We previously showed that expression of the bacterial proton pump bacterio-opsin (bO) in transgenic plants resulted in the spontaneous activation of the HR. Here we show that the bO protein is likely localised to the PM in transgenic tobacco plants. Furthermore, mutational analysis shows that induction of the HR by bO expression is dependent upon the capability of bO to translocate protons. Although bO functions as a light-driven proton pump in Halobacteria when assembled with retinal, we also show by mutational analysis that this chromophore binding is unnecessary for its in planta activity. Taken together, our results suggest that expression of bO in plants leads to the insertion of a passive proton channel into the PM. The activity of this channel in the PM results in spontaneous activation of cell death and HR-associated phenotypes including enhanced resistance to a broad spectrum of plant pathogens. Our work provides direct molecular evidence to support a working model in which alterations in ionic homeostasis at the level of the PM may work as one of the critical steps in the signalling pathway for the activation of the HR.     

Novel crosstalk between ERK MAPK and p38 MAPK leads to homocysteine‐NMDA receptor‐mediated neuronal cell death

Hyperhomocysteinemia is an independent risk factor for both acute and chronic neurological disorders, but little is known about the underlying mechanisms by which elevated homocysteine can promote neuronal cell death. We recently established a role for NMDA receptor‐mediated activation of extracellular signal‐regulated kinase (ERK)‐MAPK in homocysteine‐induced neuronal cell death. In this study, we examined the involvement of the stress‐induced MAPK, p38 in homocysteine‐induced neuronal cell death, and further explored the relationship between the two MAPKs, ERK and p38, in triggering cell death. Homocysteine‐mediated NMDA receptor stimulation and subsequent Ca²⁺ influx led to a biphasic activation of p38 MAPK characterized by an initial rapid, but transient activation followed by a delayed and more prolonged response. Selective inhibition of the delayed p38 MAPK activity was sufficient to attenuate homocysteine‐induced neuronal cell death. Using pharmacological and RNAi approaches, we further demonstrated that both the initial and delayed activation of p38 MAPK is downstream of, and dependent on activation of ERK MAPK. Our findings highlight a novel interplay between ERK and p38 MAPK in homocysteine‐NMDA receptor‐induced neuronal cell death.