ABSTRACT
Programmed cell death (PCD) is fundamentally important for plant development, abiotic stress responses and immunity, but our understanding of its regulation remains fragmented. Building a stronger research community is required to accelerate progress in this area through knowledge exchange and constructive debate. In this Viewpoint, we aim to initiate a collective effort to integrate data across a diverse set of experimental models to facilitate characterisation of the fundamental mechanisms underlying plant PCD and ultimately aid the development of a new plant cell death classification system in the future. We also put forward our vision for the next decade of plant PCD research stemming from discussions held during the 31st New Phytologist workshop, 'The Life and Death Decisions of Plant Cells' that took place at University College Dublin in Ireland (14-15 June 2023). We convey the key areas of significant progress and possible future research directions identified, including resolving the spatiotemporal control of cell death, isolation of its molecular and genetic regulators, and harnessing technical advances for studying PCD events in plants. Further, we review the breadth of potential impacts of plant PCD research and highlight the promising new applications of findings from this dynamically evolving field.
Subject(s)
Apoptosis , Research , Plants , Plant Cells/physiologyABSTRACT
Plant proteases are essential enzymes that play key roles during crucial phases of plant life. Some proteases are mainly involved in general protein turnover and recycle amino acids for protein synthesis. Other proteases are involved in cell signalling, cleave specific substrates and are key players during important genetically controlled molecular processes. Cathepsin B is a cysteine protease that can do both because of its exopeptidase and endopeptidase activities. Animal cathepsin B has been investigated for many years, and much is known about its mode of action and substrate preferences, but much remains to be discovered about this potent protease in plants. Cathepsin B is involved in plant development, germination, senescence, microspore embryogenesis, pathogen defence and responses to abiotic stress, including programmed cell death. This review discusses the structural features, the activity of the enzyme and the differences between the plant and animal forms. We discuss its maturation and subcellular localisation and provide a detailed overview of the involvement of cathepsin B in important plant life processes. A greater understanding of the cell signalling processes involving cathepsin B is needed for applied discoveries in plant biotechnology.
ABSTRACT
The Plastid Terminal Oxidase (PTOX) is a chloroplast localized plastoquinone oxygen oxidoreductase suggested to have the potential to act as a photoprotective safety valve for photosynthesis. However, PTOX overexpression in plants has been unsuccessful at inducing photoprotection, and the factors that control its activity remain elusive. Here, we show that significant PTOX activity is induced in response to high light in the model species Eutrema salsugineum and Arabidopsis thaliana. This activation correlates with structural reorganization of the thylakoid membrane. Over-expression of PTOX in mutants of Arabidopsis thaliana perturbed in thylakoid stacking also results in such activity, in contrast to wild type plants with normal granal structure. Further, PTOX activation protects against photoinhibition of Photosystem II and reduces reactive oxygen production under stress conditions. We conclude that structural re-arrangements of the thylakoid membranes, bringing Photosystem II and PTOX into proximity, are both required and sufficient for PTOX to act as a Photosystem II sink and play a role in photoprotection.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Oxidoreductases , Arabidopsis/genetics , Arabidopsis/metabolism , Electron Transport/physiology , Oxidoreductases/genetics , Oxidoreductases/metabolism , Oxygen , Photosystem II Protein Complex/genetics , Plants/metabolism , Plastids/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolismABSTRACT
Metacaspases are a class of proteases found in plants that have gained attention in recent years due to their involvement in programmed cell death (PCD) and other essential cellular processes. Although structurally homologous to caspases found in animals, metacaspases have distinct properties and functions. There are nine metacaspase genes in the Arabidopsis thaliana genome; these can be type I or type II, and working out the function of each member of the gene family is challenging. In this study, we report the characterisation of one Arabidopsis type II metacaspase, metacaspase-5 (AtMC5; AtMCA-IIb). We detected the expression of AtMC5 only under specific conditions with a strong upregulation by ER stress and oxidative stress at a narrow 6 h time point. Recombinant AtMC5 was successfully purified from E. coli, with the recombinant AtMC5 working optimally at pH 7, using an optimised reaction buffer containing 10 mM calcium chloride together with 15% sucrose. Like other metacaspases, AtMC5 cleaved after arginine residue and demonstrated a substrate preference towards VRPR. Additionally, AtMC5-RFP was shown to be localised in the cytosol and nucleus of transfected cells. We found no evidence of a strong link between AtMC5 and PCD, and the data provide additional insights into the function of metacaspases in plants and will aid in future research toward further understanding their mode of action.
Subject(s)
Caspases/classification , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/classification , Plant Proteins/classification , Terminology as Topic , Animals , Caspases/chemistry , Caspases/metabolism , Consensus , Humans , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/chemistry , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/metabolism , Plant Proteins/chemistry , Plant Proteins/metabolism , Protein Conformation , Structure-Activity RelationshipABSTRACT
The genome of the model plant Arabidopsis thaliana encodes three paralogues of the papain-like cysteine proteinase cathepsin B (AtCathB1, AtCathB2 and AtCathB3), whose individual functions are still largely unknown. Here we show that a mutated splice site causes severe truncations of the AtCathB1 polypeptide, rendering it catalytically incompetent. By contrast, AtCathB2 and AtCathB3 are effective proteases which display comparable hydrolytic properties and share most of their substrate specificities. Site-directed mutagenesis experiments demonstrated that a single amino acid substitution (Gly336âGlu) is sufficient to confer AtCathB2 with the capacity to tolerate arginine in its specificity-determining S2 subsite, which is otherwise a hallmark of AtCathB3-mediated cleavages. A degradomics approach utilizing proteome-derived peptide libraries revealed that both enzymes are capable of acting as endopeptidases and exopeptidases, releasing dipeptides from the C-termini of substrates. Mutation of the carboxydipeptidase determinant His207 also affected the activity of AtCathB2 towards non-exopeptidase substrates, highlighting mechanistic differences between plant and human cathepsin B. This was also noted in molecular modeling studies which indicate that the occluding loop defining the dual enzymatic character of cathepsin B does not obstruct the active-site cleft of AtCathB2 to the same extent as in its mammalian orthologues.
Subject(s)
Arabidopsis/enzymology , Carboxypeptidases/metabolism , Cathepsin B/metabolism , Endopeptidases/metabolism , Animals , Carboxypeptidases/chemistry , Carboxypeptidases/genetics , Cathepsin B/chemistry , Cathepsin B/genetics , Cloning, Molecular , Endopeptidases/chemistry , Endopeptidases/genetics , Models, Molecular , Mutagenesis, Site-Directed , Plant Leaves/enzymology , Real-Time Polymerase Chain Reaction , Spodoptera/cytology , Spodoptera/geneticsABSTRACT
The oligosaccharyltransferase (OT) complex catalyzes N-glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known about the structure and function of plant OT complexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified Arabidopsis OT complexes using the tandem affinity-tagged OT subunit STAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super-expression platform. Mass-spectrometry analysis of the purified complexes identified three essential OT subunits, OLIGOSACCHARYLTRANSFERASE1 (OST1), HAPLESS6 (HAP6), DEFECTIVE GLYCOSYLATION1 (DGL1), and a number of ribosomal subunits. Transmission-electron microscopy showed that STT3a becomes incorporated into OT-ribosome super-complexes formed in vivo, demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwise in planta interaction analyses of individual OT subunits demonstrated that all subunits identified in animal OT complexes are conserved in Arabidopsis and physically interact with STT3a. Genetic analysis of newly established OT subunit mutants for OST1 and DEFENDER AGAINST APOTOTIC DEATH (DAD) family genes revealed that OST1 and DAD1/2 subunits are essential for the plant life cycle. However, mutations in these individual isoforms produced much milder growth/underglycosylation phenotypes than previously reported for mutations in DGL1, OST3/6 and STT3a.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Hexosyltransferases/metabolism , Membrane Proteins/metabolism , Arabidopsis/enzymology , Arabidopsis/genetics , Arabidopsis Proteins/isolation & purification , Gene Expression Regulation, Plant , Hexosyltransferases/genetics , Hexosyltransferases/isolation & purification , Mass Spectrometry , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Microscopy, Electron, Transmission , Ribosomes/enzymology , Ribosomes/metabolism , Tandem Affinity PurificationABSTRACT
Phytaspases are plant cell death-related proteases of the subtilisin-like protease family that possess an unusual aspartate cleavage specificity. Although phytaspase activity is widespread in plants, phytaspase of Arabidopsis thaliana (L.) Heynh. has escaped detection and identification thus far. Here, we show that a single gene (At4 g10540) out of 56 A. thaliana subtilisin-like protease genes encodes a phytaspase. The recombinant phytaspase was overproduced in Nicotiana benthamiana Domin leaves, isolated, and its substrate specificity and properties were characterised. At pH 5.5, at physiological mildly acidic reaction conditions, the Arabidopsis phytaspase was shown to be strictly Asp-specific. The strongly preferred cleavage motifs of the enzyme out of a panel of synthetic peptide substrates were YVAD and IETD, while the VEID-based substrate preferred by the tobacco and rice phytaspases was almost completely resistant to hydrolysis. At neutral pH, however, the Arabidopsis phytaspase could hydrolyse peptide substrates after two additional amino acid residues, His and Phe, in addition to Asp. This observation may indicate that the repertoire of Arabidopsis phytaspase targets could possibly be regulated by the conditions of the cellular environment. Similar to tobacco and rice phytaspases, the Arabidopsis enzyme was shown to accumulate in the apoplast of epidermal leaf cells. However, in stomatal cells Arabidopsis phytaspase was observed inside the cells, possibly co-localising with vacuole. Our study thus demonstrates that the Arabidopsis phytaspase possesses both important similarities with and distinctions from the already known phytaspases, and is likely to be the most divergent member of the phytaspase family.
ABSTRACT
Autophagy is essential for protein degradation, nutrient recycling, and nitrogen remobilization. Autophagy is induced during leaf ageing and in response to nitrogen starvation, and is known to play a fundamental role in nutrient recycling for remobilization and seed filling. Accordingly, ageing leaves of Arabidopsis autophagy mutants (atg) have been shown to over-accumulate proteins and peptides, possibly because of a reduced protein degradation capacity. Surprisingly, atg leaves also displayed higher protease activities. The work reported here aimed at identifying the nature of the proteases and protease activities that accumulated differentially (higher or lower) in the atg mutants. Protease identification was performed using shotgun LC-MS/MS proteome analyses and activity-based protein profiling (ABPP). The results showed that the chloroplast FTSH (FILAMENTATION TEMPERATURE SENSITIVE H) and DEG (DEGRADATION OF PERIPLASMIC PROTEINS) proteases and several extracellular serine proteases [subtilases (SBTs) and serine carboxypeptidase-like (SCPL) proteases] were less abundant in atg5 mutants. By contrast, proteasome-related proteins and cytosolic or vacuole cysteine proteases were more abundant in atg5 mutants. Rubisco degradation assays and ABPP showed that the activities of proteasome and papain-like cysteine protease were increased in atg5 mutants. Whether these proteases play a back-up role in nutrient recycling and remobilization in atg mutants or act to promote cell death is discussed in relation to their accumulation patterns in the atg5 mutant compared with the salicylic acid-depleted atg5/sid2 double-mutant, and in low nitrate compared with high nitrate conditions. Several of the proteins identified are indeed known as senescence- and stress-related proteases or as spontaneous cell-death triggering factors.
Subject(s)
Arabidopsis/physiology , Autophagy/genetics , Cysteine Proteases/genetics , Arabidopsis/genetics , Cysteine Proteases/metabolism , Mutation , Papain/metabolism , Proteasome Endopeptidase Complex/metabolismABSTRACT
Programmed cell death (PCD) induced by endoplasmic reticulum (ER) stress is implicated in various plant physiological processes, yet its mechanism is still elusive. An activation of caspase-3-like enzymatic activity was clearly demonstrated but the role of the two known plant proteases with caspase-3-like activity, cathepsin B and proteasome subunit PBA1, remains to be established. Both genetic downregulation and chemical inhibition were used to investigate the function of cathepsin B and PBA1 in ER-stress-induced PCD (ERSID). Transcript level and activity labelling of cathepsin B were used to assess activation. To study tonoplast rupture, a plant PCD feature, both confocal and electronic microscopies were used. Cathepsin B downregulation reduced reactive oxygen species (ROS) accumulation and ERSID without affecting the induction of the unfolded protein response (UPR), but downregulation of PBA1 increased UPR and ERSID. Tonoplast rupture was not altered in the cathepsin B mutant and cathepsin B activation was independent of vacuolar processing enzyme (VPE). VPE activity was independent of cathepsin B. ERSID is regulated positively by cathepsin B and negatively by PBA1, revealing a complex picture behind caspase-3-like activity in plants. Cathepsin B may execute its function after tonoplast rupture and works in parallel with VPE.
Subject(s)
Apoptosis , Arabidopsis Proteins/metabolism , Arabidopsis/cytology , Arabidopsis/enzymology , Caspase 3/metabolism , Cathepsin B/metabolism , Cysteine Endopeptidases/metabolism , Endoplasmic Reticulum Stress , Proteasome Endopeptidase Complex/metabolism , Down-Regulation , Phenotype , Seedlings/metabolism , Unfolded Protein Response , Vacuoles/metabolism , Vacuoles/ultrastructureABSTRACT
Programmed cell death (PCD) is a critical component of plant development, defense against invading pathogens, and response to environmental stresses. In this chapter, we provide detailed technical methods for studying PCD associated with plant development or induced by abiotic stress. A root hair assay or electrolyte leakage assay are excellent techniques for the quantitative determination of PCD and/or cellular injury induced in response to abiotic stress, whereas the lace plant provides a unique model that facilitates the study of genetically regulated PCD during leaf development.
Subject(s)
Alismataceae/cytology , Cell Death , Molecular Imaging/methods , Plant Leaves/cytology , Stress, Physiological , Alismataceae/physiology , Plant Leaves/physiology , Plant Proteins/metabolism , Staining and Labeling/methodsABSTRACT
Plant cells, like cells from other kingdoms, have the ability to self-destruct in a genetically controlled manner. This process is defined as Programmed cell death (PCD). PCD can be triggered by various stimuli in plants including by endoplasmic reticulum (ER) stress. Research in the past two decades discovered that disruption of protein homeostasis in the ER could cause ER stress, which when prolonged/unresolved leads cells into PCD. ER stress-induced PCD is part of several plant processes, for instance, drought and heat stress have been found to elicit ER stress-induced PCD. Despite the importance of ER stress-induced PCD in plants, its regulation remains largely unknown, when compared with its counterpart in animal cells. In mammalian cells, several pro-apoptotic proteases called caspases were found to play a crucial role in ER stress-induced PCD. Over the past decade, several key proteases with caspase-like enzymatic activity have been discovered in plants and implicated in PCD regulation. This review covers what is known about caspase-like enzymatic activities during plant ER stress-induced PCD and discusses possible regulation pathways leading to the activation of relevant proteases in plants.
ABSTRACT
In Arabidopsis thaliana we demonstrate that dying root hairs provide an easy and rapid in vivo model for the morphological identification of apoptotic-like programmed cell death (AL-PCD) in plants. The model described here is transferable between species, can be used to investigate rates of AL-PCD in response to various treatments and to identify modulation of AL-PCD rates in mutant/transgenic plant lines facilitating rapid screening of mutant populations in order to identify genes involved in AL-PCD regulation.
ABSTRACT
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 KODâ·GFP fusion, however, localized in the cytosol of cells and not mitochondria.
Subject(s)
Apoptosis , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Peptides/metabolism , Arabidopsis Proteins/genetics , Hot Temperature , Membrane Potential, Mitochondrial , Mitochondrial Membranes/physiology , Mutant Proteins/genetics , Mutant Proteins/metabolism , Peptides/genetics , Plant Leaves/metabolism , Seedlings/metabolismABSTRACT
BACKGROUND: Programmed cell death (PCD) is an essential process for the life cycle of all multicellular organisms. In higher plants however, relatively little is known about the cascade of genes and signalling molecules responsible for the initiation and execution of PCD. To aid with the discovery and analysis of plant PCD regulators, we have designed a novel cell death assay based on low cytosolic pH as a marker of PCD. RESULTS: The acidification that occurs in the cytosol during plant PCD was monitored by way of the extinction of YFP fluorescence at low pH. This fluorescence was recovered experimentally when bringing the intracellular pH back to 7, demonstrating that there was no protein degradation of YFP. Because it uses YFP, the assay is none-destructive, does not interfere with the PCD process and allows time-lapse studies to be carried out. In addition, changes of sub-cellular localisation can be visualised during PCD using the protein of interest fused to RFP. Coupled to a transient expression system, this pH-based assay can be used to functionally analyse genes involved in PCD, using point mutations or co-expressing PCD regulators. Transfecting mBAX and AtBI-1in onion epidermal cells showed that the pH shift is downstream of PCD suppression by AtBI-1. In addition, this method can be used to score PCD in tissues of stably transformed transgenic lines. As proof of principle, we show the example of YFP extinction during xylogenesis in Arabidopsis. This demonstrates that the assay is applicable to PCD studies in a variety of tissues. CONCLUSIONS: The observation that YFP fluorescence is lost during the plant PCD process provides a new tool to study the genetic regulation and cell biology of the process. In addition, plant cell biologists should make a note of this effect of PCD on YFP fluorescence to avoid misinterpretation of their data and to select a pH insensitive reporter if appropriate. This method represents an efficient and streamlined tool expected to bring insights on the process leading to the pH shift occurring during PCD.
ABSTRACT
We report on the characterization of the interaction between reactive oxygen species signalling and abscisic acid (ABA)-mediated gene network in ozone (O(3)) stress response. To identify the stress-related signalling pathways and possible cross-talk controlled by an ABA-negative regulator, the protein phosphatase 2C abscisic acid insensitive1 (ABI1), we performed a genome-wide transcription profiling of O(3)-treated wild-type and ABI1 knockout (abi1td) plants. In addition, to better understand ABA signalling and the interactions between stress response pathways, we performed a microarray analysis of drought-treated plants. Functional categorization of the identified genes showed that ABI1 is involved in the modulation of several cellular processes including metabolism, transport, development, information pathways and variant splicing. Comparisons with available transcriptome data sets revealed the extent of ABI1 involvement in both ABA-dependent and ABA-independent gene expression. Furthermore, in O(3) stress the ABA hypersensitivity of abi1td resulted in a significant reduction of the ABA level, ethylene (ET) over-production and O(3) tolerance. Moreover, the physical interaction of ABI1 with ACC synthase2 and ACC synthase6 was shown. We provide a model explaining how ABI1 can regulate both ABA and ET biosynthesis. Altogether, our findings indicate that ABI1 plays the role of a general signal transducer linking ABA and ET biosynthesis as well as signalling pathways to O(3) stress tolerance.
Subject(s)
Abscisic Acid/biosynthesis , Arabidopsis Proteins/genetics , Arabidopsis/genetics , Ethylenes/biosynthesis , Gene Expression Profiling , Mutagenesis, Insertional/drug effects , Ozone/pharmacology , Phosphoprotein Phosphatases/genetics , Alternative Splicing/drug effects , Alternative Splicing/genetics , Arabidopsis/drug effects , Arabidopsis Proteins/metabolism , Biological Transport/drug effects , Cyclopentanes/pharmacology , Droughts , Gene Expression Regulation, Plant/drug effects , Mutation/genetics , Oxylipins/pharmacology , Phenotype , Phosphoprotein Phosphatases/metabolism , Protein Phosphatase 2C , Regulon/genetics , Reproducibility of Results , Signal Transduction/drug effects , Signal Transduction/genetics , Stress, Physiological/drug effects , Transcription, Genetic/drug effectsABSTRACT
Photosynthetic electron transport can involve either a linear flow from water to NADP, via Photosystems (PS) II and I or a cyclic flow just involving PSI. Little is known about factors regulating the relative flow through each of these pathways. We have examined photosynthetic electron transport through each system in plants of Arabidopsis thaliana in which either the PSI-D1 or PSI-E1 subunits of PSI have been knocked out. In both cases, this results in an imbalance in the turnover of PSI and PSII, such that PSII electron transport is limited by PSI turnover. Phosphorylation of light-harvesting complex II (LHCII) and its migration to PSI is enhanced but only partially reversible and not sufficient to balance photosystem turnover. In spite of this, cyclic electron flow is able to compete efficiently with PSI across a range of conditions. In dark-adapted leaves, the efficiency of cyclic relative to linear flow induced by far-red light is increased, implying that the limiting step of cyclic flow lies in the re-injection of electrons into the electron transport chain. Illumination of leaves with white light resulted in transient induction of a significant non-photochemical quenching in knockout plants which is probably high energy state quenching induced by cyclic electron flow. At high light and at low CO(2), non-photochemical quenching was greater in the knockout plants than in the wildtype. Comparison of PSI and PSII turnover under such conditions suggested that this is generated by cyclic electron flow around PSI. We conclude that, when the concentration of PSI is limiting, cyclic electron flow is still able to compete effectively with linear flow to maintain a high DeltapH to regulate photosynthesis.
Subject(s)
Arabidopsis/metabolism , Photosystem I Protein Complex/metabolism , Photosystem II Protein Complex/metabolism , Arabidopsis/radiation effects , Arabidopsis Proteins , Carbon Dioxide/metabolism , Chlorophyll/metabolism , Electron Transport/radiation effects , Fluorescence , Light , Light-Harvesting Protein Complexes/metabolism , Membrane Potentials/radiation effects , Mutation/genetics , Phosphorylation/radiation effects , Photosystem I Protein Complex/genetics , Plant Leaves/metabolism , Plant Leaves/radiation effects , Protein Transport/radiation effects , Thylakoids/metabolism , Thylakoids/radiation effectsABSTRACT
Promoter trapping is a particular gene trap strategy that represents a valuable tool for the discovery of specific cell-type markers. The principle is to generate a collection of transgenic lines with random insertions of a promoter-less reporter gene and to screen for specific reporter activity in the domain of interest. The use of beta-glucuronidase (GUS) as a reporter gene provides a simple and sensitive assay that allows identification of very restricted expression patterns and makes the promoter trap appropriate to study embryogenesis. Plant embryogenesis starts at the fertilization of the egg cell encapsulated in the maternal tissue and leads to the establishment of a new organism capable of an autonomous life. Uncovering genes specifically expressed in sub-domain of the embryo during its development represents a major technical challenge due, in part, to size and accessibility limitations. Promoter trapping approaches have been successfully used to overcome these problems. The trapped activity represents thereafter a useful genetic marker of the uncovered cell type, which is expected to reveal the properties of a specific promoter shedding light on a new gene function. In this chapter, protocols for examining and documenting GUS reporter gene activities in the embryo are described. Methods for the amplification of sequences flanking insertions and subsequent molecular and genetic characterization are provided.
Subject(s)
Arabidopsis/embryology , Arabidopsis/genetics , Glucuronidase/genetics , Promoter Regions, Genetic/genetics , Seeds/physiology , DNA Primers , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Genes, Reporter , Polymerase Chain Reaction/methodsABSTRACT
When plants experience an imbalance between the absorption of light energy and the use of that energy to drive metabolism, they are liable to suffer from oxidative stress. Such imbalances arise due to environmental conditions (e.g. heat, chilling or drought), and can result in the production of reactive oxygen species (ROS). Here, we present evidence for a novel protective process - feedback redox regulation via the redox poise of the NADP(H) pool. Photosynthetic electron transport was studied in two transgenic tobacco (Nicotiana tabacum) lines - one having reduced levels of ferredoxin NADP+-reductase (FNR), the enzyme responsible for reducing NADP+, and the other reduced levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the principal consumer of NADPH. Both had a similar degree of inhibition of carbon fixation and impaired electron transport. However, whilst FNR antisense plants were obviously stressed, with extensive bleaching of leaves, GAPDH antisense plants showed no visible signs of stress, beyond having a slowed growth rate. Examination of electron transport in these plants indicated that this difference is due to feedback regulation occurring in the GAPDH but not the FNR antisense plants. We propose that this reflects the occurrence of a previously undescribed regulatory pathway responding to the redox poise of the NADP(H) pool.
Subject(s)
NADP/metabolism , Nicotiana/metabolism , Photosynthesis/physiology , Electron Transport/physiology , Ferredoxin-NADP Reductase/genetics , Ferredoxin-NADP Reductase/metabolism , Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)/genetics , Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)/metabolism , Oxidation-Reduction , Plants, Genetically Modified , Nicotiana/genetics , Nicotiana/physiologyABSTRACT
The extent of conservation in the programmed cell death pathways that are activated in species belonging to different kingdoms is not clear. Caspases are key components of animal apoptosis; caspase activities are detected in both animal and plant cells. Yet, while animals have caspase genes, plants do not have orthologous sequences in their genomes. It is 10 years since the first caspase activity was reported in plants, and there are now at least eight caspase activities that have been measured in plant extracts using caspase substrates. Various caspase inhibitors can block many forms of plant programmed cell death, suggesting that caspase-like activities are required for completion of the process. Since plant metacaspases do not have caspase activities, a major challenge is to identify the plant proteases that are responsible for the caspase-like activities and to understand how they relate, if at all, to animal caspases. The protease vacuolar processing enzyme, a legumain, is responsible for the cleavage of caspase-1 synthetic substrate in plant extracts. Saspase, a serine protease, cleaves caspase-8 and some caspase-6 synthetic substrates. Possible scenarios that could explain why plants have caspase activities without caspases are discussed.