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Extracellular ATP is a purinergic signal with important functions in regulating plant growth and stress-adaptive responses, including programmed cell death. While signalling events proximate to receptor activation at the plasma membrane have been characterised, downstream protein targets and the mechanism of cell death activation/regulation are unknown. We designed a proteomic screen to identify ATP-responsive proteins in Arabidopsis cell cultures exposed to mycotoxin stress via fumonisin B1 (FB1) application. Arabidopsis RIBONUCLEASE 1 (RNS1) was identified by the screen and transgenic plants overexpressing native RNS1showed greater susceptibility to FB1, while a gene knockout rns1 mutant and antisense RNS1 transgenic plants were resistant to FB1-induced cell death. Native RNS1 complemented rns1 mutants and restored cell death response to FB1, while a catalytically inactive version of the ribonuclease could not. The FB1 resistance of salicylic acid-depleted nahG-expressing plants was abolished by transformation with native RNS1, but not the catalytically dead version. The mechanism of FB1-induced cell death is activation of RNS1-dependent RNA cleavage, which is blocked by ATP via RNS1 suppression, or enhanced by salicylic acid (SA) through induction of RNS1 expression. Our study reveals RNS1 as a previously unknown convergence point of ATP and SA signalling in the regulation of stress-induced cell death.

When exposed to drought stress many plants reprogram their gene expression to activate adaptive biochemical and physiological responses for survival. However, most of the well-studied adaptive responses are common between drought-sensitive and drought-tolerant species, making it difficult to identify the key mechanisms underpinning successful drought tolerance in crops. We developed a sorghum experimental system that compares between drought-sensitive (ICSB338) and enhanced drought-tolerant (SA1441) varieties. We show that sorghum activates a swift and robust stomatal shutdown to preserve leaf water content when water stress has been sensed. Water uptake is enhanced via increasing root cell water potential through the rapid biosynthesis of predominantly glycine betaine and an increased root-to-shoot ratio to explore more soil volume for water. In addition to stomatal responses, there is a prompt accumulation of proline in leaves and effective protection of chlorophyll during periods of water limitation. Root and stomatal functions rapidly recover from water limitation (within 24 h of re-watering) in the drought-tolerant variety, but recovery is impaired in the drought-sensitive sorghum variety. Analysis of the root proteome revealed complex protein networks that possibly underpin sorghum responses to water limitation. Common and unique protein changes between the two sorghum varieties provide new targets for future use in investigating sorghum drought tolerance.

Fossil‐fuel processing and consumption contaminates air, soil, and water resources through the release of hazardous chemicals. The harnessing of renewable energy resources and development of sustainable technologies have become prime targets of research and increased investment to protect the environment. The use of bio‐based feedstocks in energy production provides a valuable pollution‐curbing pathway with sustainability credentials, especially when wastewater is used to provide the nutrient requirements. The filamentous cyanobacterium Trichormus variabilis has attracted substantial attention from researchers due to its potential for dual industrial functions in bioenergy production and bioremediation. This species can use the power of sunlight energy efficiently to fix atmospheric CO2 and to generate valuable chemical compounds, such as carbohydrates and fatty acids, which can be converted to biofuels. As it grows in nutrient‐rich wastewater (industrial effluent) it can serve as a bio‐absorbant and replace costly chemical catalysts and nano‐materials traditionally used for the removal of nutrients and metals. However, no recent review has presented the potential for state‐of‐the‐art T. variabilis‐driven phycoremediation‐bioenergy production systems. This review suggests possible routes from phycoremediation to energy production as a strategy for developing the industrial application of T. variabilis. It brings important research results on this species together and highlights major related challenges and opportunities. It explores the current status of the use of algae in bioremediation and the production of liquid and gaseous fuels utilizing wild‐type and mutants of T. variabilis. Finally, key points underlying the potential for future research on optimization of robust technologies for supplying sustainable bioenergy using this organism are presented.

To conserve freshwater resources, domestic and industrial wastewater is recycled. Algal systems have emerged as an efficient, low-cost option for treatment (phycoremediation) of nutrient-rich wastewater and environmental protection. However, industrial wastewater may contain growth inhibitory compounds precluding algal use in phycoremediation. Therefore, extremophyte strains, which thrive in hostile environments, are sought-after. Here, we isolated such an alga - a strain of Synechocystis sp. we found to be capable of switching from commensal exploitation of the nitrogen-fixing Trichormus variabilis, for survival in nitrogen-deficient environments, to free-living growth in nitrate abundance. In nitrogen depletion, the cells are tethered to polysaccharide capsules of T. variabilis using nanotubular structures, presumably for nitrate acquisition. The composite culture failed to establish in industrial/domestic waste effluent. However, gradual exposure to increasing wastewater strength over time untethered Synechocystis cells and killed off T. variabilis. This switched the culture to a stress-acclimated monoculture of Synechocystis sp., which rapidly grew and flourished in wastewater, with ammonium and phosphate removal efficiencies of 99.4% and 97.5%, respectively. Therefore, this strain of Synechocystis sp. shows great promise for use in phycoremediation, with potential to rapidly generate biomass that can find use as a green feedstock for valuable bio-products in industrial applications.

Sorghum is a cereal crop with key agronomic traits of drought and heat stress tolerance, making it an ideal food and industrial commodity for hotter and more arid climates. These stress tolerances also present a useful scientific resource for studying the molecular basis for environmental resilience. Here we provide an extensive review of current transcriptome and proteome works conducted with laboratory, greenhouse, or field-grown sorghum plants exposed to drought, osmotic stress, or treated with the drought stress-regulatory phytohormone, abscisic acid. Large datasets from these studies reveal changes in gene/protein expression across diverse signaling and metabolic pathways. Together, the emerging patterns from these datasets reveal that the overall functional classes of stress-responsive genes/proteins within sorghum are similar to those observed in equivalent studies of other drought-sensitive model species. This highlights a monumental challenge of distinguishing key regulatory genes/proteins, with a primary role in sorghum adaptation to drought, from genes/proteins that change in expression because of stress. Finally, we discuss possible options for taking the research forward. Successful exploitation of sorghum research for implementation in other crops may be critical in establishing climate-resilient agriculture for future food security.

ATP is secreted to the extracellular matrix, where it activates plasma membrane receptors for controlling plant growth and stress-adaptive processes. DOES NOT RESPOND TO NUCLEOTIDES 1 (DORN1), was the first plant ATP receptor to be identified but key downstream proteins remain sought after. Here, we identified 120 proteins secreted by Arabidopsis cell cultures and screened them for putative stress-responsive proteins using ATP-affinity purification. We report three Arabidopsis proteins isolated by ATP-affinity: PEROXIDASE 52, SUBTILASE-LIKE SERINE PROTEASE 1.7 and PHOSPHOLIPASE C-LIKE 1. In wild-type Arabidopsis, the expression of genes encoding all three proteins responded to fumonisin B1, a cell death-activating mycotoxin. The expression of PEROXIDASE 52 and PHOSPHOLIPASE C-LIKE 1 was altered in fumonisin B1-resistant salicylic acid induction-deficient (sid2) mutants. Exposure to fumonisin B1 suppressed PHOSPHOLIPASE C-LIKE 1 expression in sid2 mutants, suggesting that the inactivation of this gene might provide mycotoxin tolerance. Accordingly, gene knockout mutants of PHOSPHOLIPASE C-LIKE 1 were resistant to fumonisin B1-induced death. The activation of PHOSPHOLIPASE C-LIKE 1 gene expression by exogenous ATP was not blocked in dorn1 loss-of-function mutants, indicating that DORN1 is not required. Furthermore, exogenous ATP rescued both the wild type and the dorn1 mutants from fumonisin-B1 toxicity, suggesting that different ATP receptor(s) are operational in this process. Our results point to the existence of additional plant ATP receptor(s) and provide crucial downstream targets for use in designing screens to identify these receptors. Finally, PHOSPHOLIPASE C-LIKE 1 serves as a convergence point for fumonisin B1 and extracellular ATP signalling, and functions in the Arabidopsis stress response to fumonisin B1.

Water treatment residual (WTR) is a by-product of municipal clean water treatment which is normally disposed to landfill. WTR production is anticipated to increase as the demand for potable water rises, especially in sub-Saharan Africa (SSA) due to rapid population growth and urbanisation; this has driven municipalities to look for alternative ways to utilise this product and to address SDG12 by reusing wastes in the circular economy. Although significant strides have been made into research on use of WTR as a soil amendment, there are mixed reports on its efficacy at improving either soil structure or plant yield. We do know that nutrients and specifically phosphorus (P) adsorption by both the aluminium (Al) and/or iron (Fe) oxides in WTR, have a deleterious effect on plant growth. We present results on the co-application of WTR with an organic plant based compost together with inorganic P additions in a 5 week greenhouse maize pot trial. There were no significant differences in maize growth and biomass production between the coamendment and sole compost amendments at P < 0.05. Addition of P further increased maize dry matter yield two fold in the co-amendment, a 403% increase compared to the unamended control. Preliminary findings show that WTR can be co-applied with another organic nutrient resource without incurring a yield penalty and thus could be a sustainable option for reusing WTR to enhance environmental protection.

Despite having a network of cytoplasmic interconnections (plasmodesmata) facilitating rapid exchange of metabolites and signal molecules, plant cells use the extracellular matrix as an alternative route for cell-cell communication. The need for extracellular signalling in plasmodesmata-networked tissues is baffling. A hypothesis is proposed that this phenomenon defines the plant extracellular matrix as a 'democratic space' for collective decision-making in a decentralized system, similar to quorum-sensing in bacteria. Extracellular communication enables signal integration and coordination across several cell layers through ligand-activated plasma membrane receptors. Recent results from drought stress-adaptive responses and light-mediated signalling in cell death activation show operational utility of this decision-making process. Opportunities are discussed for new innovations in drought gene discovery using platforms targeting the extracellular matrix.

Emergence of glyphosate-resistant horseweed (Conyza canadensis) biotypes is an example of how unrelenting use of a single mode of action herbicide in agricultural weed control drives genetic adaptation in targeted species. While in other weeds glyphosate resistance arose from target site mutation or target gene amplification, the resistance mechanism in horseweed uses neither of these, being instead linked to reduced herbicide uptake and/or translocation. The molecular components underpinning horseweed glyphosate-resistance remain unknown. Here, we used an in vitro leaf disc system for comparative analysis of proteins extracted from control and glyphosate-treated tissues of glyphosate-resistant and glyphosate-susceptible biotypes. Analysis of shikimic acid accumulation, ABC-transporter gene expression, and cell death were used to select a suitable glyphosate concentration and sampling time for enriching proteins pivotal to glyphosate resistance. Protein gel analysis and mass spectrometry identified mainly chloroplast proteins differentially expressed between the biotypes before and after glyphosate treatment. Chloroplasts are the organelles in which the shikimate pathway, which is targeted by glyphosate, is located. Calvin cycle enzymes and proteins of unknown function were among the proteins identified. Our study provides candidate proteins that could be pivotal in engendering resistance and implicates chloroplasts as the primary sites driving glyphosate-resistance in horseweed.

Light plays an important role in plant growth, development, and response to environmental stresses. To investigate the effects of light on the plant responses to cadmium (Cd) stress, we performed a comparative physiological and proteomic analysis of light- and dark-grown Arabidopsis cells after exposure to Cd. Treatment with different concentrations of Cd resulted in stress-related phenotypes such as cell growth inhibition and decline of cell viability. Notably, light-grown cells were more sensitive to heavy metal toxicity than dark-grown cells, and the basis for this appears to be the elevated Cd accumulation, which is twice as much under light than dark growth conditions. Protein profiles analyzed by 2D DIGE revealed a total of 162 protein spots significantly changing in abundance in response to Cd under at least one of these two growing conditions. One hundred and ten of these differentially expressed protein spots were positively identified by MS/MS and they are involved in multiple cellular responses and metabolic pathways. Sulfur metabolism-related proteins increased in relative abundance both in light- and dark-grown cells after exposure to Cd. Proteins involved in carbohydrate metabolism, redox homeostasis, and anti-oxidative processes were decreased both in light- and dark-grown cells, with the decrease being lower in the latter case. Remarkably, proteins associated with cell wall biosynthesis, protein folding, and degradation showed a light-dependent response to Cd stress, with the expression level increased in darkness but suppressed in light. The possible biological importance of these changes is discussed.

Complex signalling systems have evolved in multicellular organisms to enable cell-to-cell communication during growth and development. In plants, cell communication via the extracellular matrix (apoplast) controls many processes vital for plant survival. Secretion of ATP into the extracellular matrix is now recognised as a previously unknown stimulus for cell signalling with a role in many aspects of plant physiology. In the last decade, the secondary messenger molecules in extracellular ATP signalling were identified, but the downstream gene and protein networks that underpin plant responses to extracellular ATP are only beginning to be characterised. Here we review the current status of our knowledge of plant extracellular signalling and demonstrate how applying state-of-the art proteomic technologies is rapidly bringing new discoveries in extracellular ATP research. We discuss how monitoring of the global proteomic profile during responses to modulation of extracellular ATP signalling has led to novel insight into pathogen defence systems and plant programmed cell death regulation. On the basis of extensive proteomic, pharmacological, and reverse genetics data, extracellular ATP has been confirmed to constitute an important molecular switch that tightly controls organellar energy metabolism, reprogramming of primary metabolic pathways, and redirection of resources to protein networks that support adaptation of plants to stress.

Extracellular adenosine 5'-triphosphate (eATP) is emerging as an important plant signalling compound capable of mobilising intracellular second messengers such as Ca(2+), nitric oxide, and reactive oxygen species. However, the downstream molecular targets and the spectrum of physiological processes that eATP regulates are largely unknown. We used exogenous ATP and a non-hydrolysable analogue as probes to identify the molecular and physiological effects of eATP-mediated signalling in tobacco. 2-DE coupled with MS/MS analysis revealed differential protein expression in response to perturbation of eATP signalling. These proteins are in several functional classes that included photosynthesis, mitochondrial ATP synthesis, and defence against oxidative stress, but the biggest response was in the pathogen defence-related proteins. Consistent with this, impairment of eATP signalling induced resistance against the bacterial pathogen Erwinia carotovora subsp. carotovora. In addition, disease resistance activated by a fungal pathogen elicitor (xylanase from Trichoderma viride) was concomitant with eATP depletion. These results reveal several previously unknown putative molecular targets of eATP signalling, which pinpoint eATP as an important hub at which regulatory signals of some major primary metabolic pathways and defence responses are integrated.

Extracellular ATP regulates higher plant growth and adaptation. The signalling events may be unique to higher plants, as they lack animal purinoceptor homologues. Although it is known that plant cytosolic free Ca2+ can be elevated by extracellular ATP, the mechanism is unknown. Here, we have studied roots of Arabidopsis thaliana to determine the events that lead to the transcriptional stress response evoked by extracellular ATP. Root cell protoplasts were used to demonstrate that signalling to elevate cytosolic free Ca2+ is determined by ATP perception at the plasma membrane, and not at the cell wall. Imaging revealed that extracellular ATP causes the production of reactive oxygen species in intact roots, with the plasma membrane NADPH oxidase AtRBOHC being the major contributor. This resulted in the stimulation of plasma membrane Ca2+-permeable channels (determined using patch-clamp electrophysiology), which contribute to the elevation of cytosolic free Ca2+. Disruption of this pathway in the AtrbohC mutant impaired the extracellular ATP-induced increase in reactive oxygen species (ROS), the activation of Ca2+ channels, and the transcription of the MAP kinase3 gene that is known to be involved in stress responses. This study shows that higher plants, although bereft of purinoceptor homologues, could have evolved a distinct mechanism to transduce the ATP signal at the plasma membrane.

Slow progress has been made in discovering plant genes governing the interaction of plant pathogens and their hosts using classical genetic approaches. Extensive studies employing DNA microarray techniques to identify global changes in gene expression during pathogen– host interaction have greatly enhanced discovery of genetic components regulating the plant defence response to pathogen attack. In this study, a complementary approach was used to identify changes in protein abundance during interaction of Arabidopsis cell cultures with a pathogen-derived elicitor. The soluble protein fractions were analysed by two-dimensional difference gel electrophoresis and proteins differentially expressed in response to treatment with fungal elicitor were identified via matrix-assisted laser desorption ionization–time of flight mass spectrometry. Elicitor responsive proteins included molecular chaperones, oxidative stress defence proteins, mitochondrial proteins, and enzymes of a diverse number of metabolic pathways. The findings, in combination with currently available microarray data, will form the basis of a filter to identify pivotal genes whose role in pathogen defence systems will require confirmation using gene knockout mutants.

The extracellular matrix is a vital compartment in plants with a prominent role in defence against pathogen attack. Using a maize cell suspension culture system and pathogen elicitors, responses to pathogen attack that are localised to the extracellular matrix were examined by a proteomic approach. Elicitor treatment of cell cultures induced a rapid change in the phosphorylation status of extracellular peroxidases, the apparent disappearance of a putative extracellular beta-N-acetylglucosamonidase, and accumulation of a secreted putative xylanase inhibitor protein. Onset of the defence response was attended by an accumulation of glyceraldehyde-3-phosphate dehydrogenase and a fragment of a putative heat shock protein. Several distinct spots of both proteins, which preferentially accumulated in cell wall protein fractions, were identified. These three novel observations, viz. (i) secretion of a new class of putative enzyme inhibitor, (ii) the apparent recruitment of classical cytosolic proteins into the cell wall and (ii) the change in phosphorylation status of extracellular matrix proteins, suggest that the extracellular matrix plays a complex role in defence. We discuss the role of the extracellular matrix in signal modulation during pathogen-induced defence responses.

Arabidopsis thaliana cell suspension cultures have been used to investigate the effects of salinity and hyperosmotic stress on plant cellular proteins. We show that 200 mM NaCl and 400 mM sorbitol treatments induce extracellular medium acidification in Arabidopsis cell cultures, a typical response of plant cells to salt and hyperosmotic stress. Using (35)S-labelled amino acids, we demonstrated that NaCl causes a transient suppression of de novo protein synthesis, from which the cells recover within 4 h. Changes in the abundance of cellular proteins 6 h post NaCl and sorbitol treatments were analysed by 2-DE. Of a total of 2,949 protein spots detected on the gels, 266 showed significant changes in abundance across five independent experiments. Using MALDI-TOF MS, we identified 75 salt and sorbitol responsive spots. These fall into 10 functional categories that include H(+) transporting ATPases, signal transduction related proteins, transcription/translation related proteins, detoxifying enzymes, amino acid and purine biosynthesis related proteins, proteolytic enzymes, heat-shock proteins, carbohydrate metabolism-associated proteins and proteins with no known biological functions.

We initiated a proteomic study as part of a programme aimed at discovering novel functions of the plant cell wall. Cell-wall fragments isolated from cell-suspension cultures of Arabidopsis thaliana were stripped of protein sequentially using CaCl2 and a urea-based buffer. The protein fractions were separated by two-dimensional gel electrophoresis and individual proteins were identified by MS. We identified a number of proteins considered to be resident in other organelles but not the cell wall on the basis of their classical biological function. These included citrate synthase, which is known to be targeted to mitochondria, peroxisomes and glyoxysomes, and luminal binding protein, which is an ER (endoplasmic reticulum)-resident protein. Searches of the Arabidopsis database revealed that there are several genes encoding putative citrate synthase and luminal binding protein. We have also performed detailed analyses of the protein sequences and this paper shows how each one contains encrypted targeting information that results in the export of the protein to the extracellular matrix. We discuss the presence of alternative non-classical secretory pathways in plants.

A proteomic approach has been applied to investigate changes in the extracellular matrix of Arabidopsis thaliana cell suspension cultures following treatments with two fungal pathogen elicitors, chitosan and extracts of Fusarium moniliforme. The oxidative burst and induction of glutathione S-transferase were used as markers for induction of the pathogen defence response. Changes in the cell wall and culture filtrate proteome were profiled. Proteins whose abundance changed reproducibly were analysed via matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS). An increase in the level of two classical cell wall proteins (a putative endochitinase and a polygalacturonase inhibiting protein) and two novel proteins (a putative receptor-like protein kinase and a probable apospory-associated protein) were seen at 24 hours following elicitation. The level of an unknown protein and a hypothetical protein, which has some homology to serine carboxypeptidases, were decreased at 24 hours post-elicitation. In the culture filtrate extracts, we identified two pathogen elicitor responsive proteins, a xyloglucan endo-1,4-beta-D glucanases (XEG) and a peroxidase. Using a combination of two-dimensional polyacrylamide gel electrophoresis, immunoblotting with a phosphotyrosine-specific antibody, and MALDI-TOF MS we discovered that spots that represent putative lectin receptor-like kinase, a putative endochitinase and a XEG possess phosphorylated tyrosine residues. The identification of phosphorylated bona fide cell wall proteins and a putative extracellular receptor-like kinase with no transmembrane domain implicate the existence of an extracellular phosphorylation network which could be involved in intercellular communication.

Programmed cell death is essential for plant development and stress adaptation. A detailed understanding of the signal transduction pathways that regulate plant programmed cell death requires identification of the underpinning protein networks. Here, we have used a protagonist and antagonist of programmed cell death triggered by fumonisin B1 as probes to identify key cell death regulatory proteins in Arabidopsis. Our hypothesis was that changes in the abundance of cell death-regulatory proteins induced by the protagonist should be blocked or attenuated by concurrent treatment with the antagonist. We focused on proteins present in the mobile phase of the extracellular matrix on the basis that they are important for cell–cell communications during growth and stress-adaptive responses. Salicylic acid, a plant hormone that promotes programmed cell death, and exogenous ATP, which can block fumonisin B1-induced cell death, were used to treat Arabidopsis cell suspension cultures prior to isobaric-tagged relative and absolute quantitation analysis of secreted proteins. A total of 33 proteins, whose response to salicylic acid was suppressed by ATP, were identified as putative cell death-regulatory proteins. Among these was CYCLASE1, which was selected for further analysis using reverse genetics. Plants in which CYCLASE1 gene expression was knocked out by insertion of a transfer-DNA sequence manifested dramatically increased cell death when exposed to fumonisin B1 or a bacterial pathogen that triggers the defensive hypersensitive cell death. Although pathogen inoculation altered CYCLASE1 gene expression, multiplication of bacterial pathogens was indistinguishable between wild type and CYCLASE1 knockout plants. However, remarkably severe chlorosis symptoms developed on gene knockout plants in response to inoculation with either a virulent bacterial pathogen or a disabled mutant that is incapable of causing disease in wild type plants. These results show that CYCLASE1, which had no known function hitherto, is a negative regulator of cell death and regulates pathogen-induced symptom development in Arabidopsis.

Resistance genes allow plants to recognize specific pathogens. Recognition results in the activation of a variety of defence responses, including localized programmed cell death (the hypersensitive response), synthesis of pathogenesis-related proteins and induction of systemic acquired resistance. These responses are co-ordinated by a branching signal transduction pathway. In tobacco, one branch activates virus resistance, and might require the mitochondrial alternative oxidase to operate. Here we discuss the evidence for this virus-specific branch of the transduction pathway and assess what must be done to further understand virus resistance and the role of the alternative oxidase in its induction.

Cadmium is an environmental pollutant with deleterious effects on both prokaryotic and eukaryotic organisms. In plants, the effects of cadmium toxicity are concentration dependent; lower doses destabilize many physiological processes and inhibit cell growth and multiplication, while higher doses evoke a more severe response that triggers activation of cell death. We recently investigated the effects of light on cadmium toxicity in Arabidopsis using a cell suspension culture system. Although not affecting the inhibitory effects on cell multiplication, we found that light is a powerful regulator of Cd-induced cell death. A very specific proteomic response, which was clearly controlled by light, preceded cell death. Here we discuss the implications of these findings and highlight similarities between the regulation of cell death triggered by Cd and fumonisin B1. We consider how both compounds could be useful tools in dissecting plant cell death signaling.

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.

Despite intense research on light responses in plants, the consequences of a simple shift from light to darkness are largely unexplored. In this research, the physiological outcome and proteomic changes in Arabidopsis cell suspension cultures after switching from light to total darkness were examined. Deprivation of light led to a visible loss of chlorophyll and failure to develop functional chloroplasts that are present in light-grown cells. This response was accompanied by a significant increase in the cell multiplication rate, most likely due to decreased formation of the damaging reactive oxygen species in the dark. Additionally, there were significant changes in the abundance of 46 protein spots (mostly assigned to chloroplasts, mitochondria and membranes) which were resolved by two-dimensional difference gel electrophoresis and mass spectrometric analysis. All identified chloroplast proteins were down-regulated in response to sustained darkness. In contrast, all differentially expressed proteins associated with cell wall biosynthesis were up-regulated by the dark treatment. Changes in the levels of these proteins were consistent with the observed morphological and physiological changes of the cells. These results reveal a comprehensive picture of the dark response in Arabidopsis cells and provide a useful platform for further characterization of gene function and regulation in plant responses to light.

Living organisms acquire or synthesize high energy molecules, which they frugally conserve and use to meet their cellular metabolic demands. Therefore, it is surprising that ATP, the most accessible and commonly utilized chemical energy carrier, is actively secreted to the extracellular matrix of cells. It is now becoming clear that in plants this extracellular ATP (eATP) is not wasted, but harnessed at the cell surface to signal across the plasma membrane of the secreting cell and neighboring cells to control gene expression and influence plant development. Identification of the gene/protein networks regulated by eATP-mediated signaling should provide insight into the physiological roles of eATP in plants. By disrupting eATP-mediated signaling, we have identified pathogen defense genes as part of the eATP-regulated gene circuitry, leading us to the discovery that eATP is a negative regulator of pathogen defense in plants.(1) Previously, we reported that eATP is a key signal molecule that modulates programmed cell death in plants.(2) A complex picture is now emerging, in which eATP-mediated signaling cross-talks with signaling mediated by the major plant defense hormone, salicylic acid, in the regulation of pathogen defense and cell death.

ATP is a vital molecule used by living organisms as a universal source of energy required to drive the cogwheels of intracellular biochemical reactions necessary for growth and development. Animal cells release ATP to the extracellular milieu, where it functions as the primary signaling cue at the epicenter of a diverse range of physiological processes. Although recent findings revealed that intact plant tissues release ATP as well, there is no clearly defined physiological function of extracellular ATP in plants. Here, we show that extracellular ATP is essential for maintaining plant cell viability. Its removal by the cell-impermeant traps glucose-hexokinase and apyrase triggered death in both cell cultures and whole plants. Competitive exclusion of extracellular ATP from its binding sites by treatment with beta,gamma-methyleneadenosine 5'-triphosphate, a nonhydrolyzable analog of ATP, also resulted in death. The death response was observed in Arabidopsis thaliana, maize (Zea mays), bean (Phaseolus vulgaris), and tobacco (Nicotiana tabacum). Significantly, we discovered that fumonisin B1 (FB1) treatment of Arabidopsis triggered the depletion of extracellular ATP that preceded cell death and that exogenous ATP rescues Arabidopsis from FB1-induced death. These observations suggest that extracellular ATP suppresses a default death pathway in plants and that some forms of pathogen-induced cell death are mediated by the depletion of extracellular ATP.

With the completion of the Arabidopsis genome, many hypothetical proteins have been predicted without any information on their expression, subcellular localisation and function. We have performed proteomic analysis of proteins sequentially extracted from enriched Arabidopsis cell wall fractions and separated by two-dimensional gel electrophoresis (2-DE). The proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry and genomic database searches. This is part of a targeted exercise to establish the entire Arabidopsis secretome database. We report evidence for new proteins of unknown function whose existence had been predicted from genomic sequences and, furthermore, localise them to the cell wall. In addition, we observed an unexpected presence in the cell wall preparations of proteins whose known biochemical activity has never been associated with this compartment hitherto. We discuss the implications of these findings and present results suggesting a possible involvement of cell wall kinases in plant responses to pathogen attack.

Salicylhydroxamic acid (SHAM), an inhibitor of alternative oxidase (AOX), blocks salicylic acid-induced resistance to tobacco mosaic virus (TMV) but does not inhibit pathogenesis-related PR-1 protein synthesis or resistance to fungal and bacterial pathogens. We found that the synthetic resistance-inducing chemical 2, 6-dichloroisonicotinic acid also induced Aox transcript accumulation and SHAM-sensitive resistance to TMV. The respiratory inhibitors antimycin A and KCN also induced Aox transcript accumulation and resistance to TMV but did not induce PR-1 accumulation. Tobacco plants of the TMV-resistant cultivar Samsun NN transformed with the salicylic acid hydroxylase (nahG) gene could no longer restrict virus to the inoculation site, resulting in spreading necrosis instead of discrete necrotic lesions. Treatment with KCN restored TMV localization and normal lesion morphology. SHAM antagonized this effect, allowing virus escape and spreading necrosis to resume. The results demonstrate the importance of the SHAM-sensitive (potentially AOX-dependent) signal transduction pathway in mediating virus localization early in the hypersensitive response.

Extracellular ATP is an important signal molecule required to cue plant growth and developmental programs, interactions with other organisms, and responses to environmental stimuli. The molecular targets mediating the physiological effects of extracellular ATP in plants have not yet been identified. We developed a well characterized experimental system that depletes Arabidopsis cell suspension culture extracellular ATP via treatment with the cell death-inducing mycotoxin fumonisin B1. This provided a platform for protein profile comparison between extracellular ATP-depleted cells and fumonisin B1-treated cells replenished with exogenous ATP, thus enabling the identification of proteins regulated by extracellular ATP signaling. Using two-dimensional difference in-gel electrophoresis and matrix-assisted laser desorption-time of flight MS analysis of microsomal membrane and total soluble protein fractions, we identified 26 distinct proteins whose gene expression is controlled by the level of extracellular ATP. An additional 48 proteins that responded to fumonisin B1 were unaffected by extracellular ATP levels, confirming that this mycotoxin has physiological effects on Arabidopsis that are independent of its ability to trigger extracellular ATP depletion. Molecular chaperones, cellular redox control enzymes, glycolytic enzymes, and components of the cellular protein degradation machinery were among the extracellular ATP-responsive proteins. A major category of proteins highly regulated by extracellular ATP were components of ATP metabolism enzymes. We selected one of these, the mitochondrial ATP synthase β-subunit, for further analysis using reverse genetics. Plants in which the gene for this protein was knocked out by insertion of a transfer-DNA sequence became resistant to fumonisin B1-induced cell death. Therefore, in addition to its function in mitochondrial oxidative phosphorylation, our study defines a new role for ATP synthase β-subunit as a pro-cell death protein. More significantly, this protein is a novel target for extracellular ATP in its function as a key negative regulator of plant cell death.

With the completion of the Arabidopsis genome, many hypothetical proteins have been predicted without any information on their expression, subcellular localisation and function. We have performed proteomic analysis of proteins sequentially extracted from enriched Arabidopsis cell wall fractions and separated by two-dimensional gel electrophoresis (2-DE). The proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry and genomic database searches. This is part of a targeted exercise to establish the entire Arabidopsis secretome database. We report evidence for new proteins of unknown function whose existence had been predicted from genomic sequences and, furthermore, localise them to the cell wall. In addition, we observed an unexpected presence in the cell wall preparations of proteins whose known biochemical activity has never been associated with this compartment hitherto. We discuss the implications of these findings and present results suggesting a possible involvement of cell wall kinases in plant responses to pathogen attack.

Thermogenesis in Arum species is induced by salicylic acid (SA) and caused by activation of the alternative respiration pathway and the alternative oxidase (AOX), resulting in heat production. The enzymes phosphoenolpyruvate carboxylase (PEPCase) and NAD-dependent malic enzyme (NAD-ME) also show dramatic increases in activity during thermogenesis and are essential for heat generation. In this current study, we characterized the timing and localization of changes in levels of AOX, NAD-ME, and PEPCase polypeptide accumulation, and changes in Ppc and Me mRNA accumulation, in various Arum tissues during prethermogenic development and during thermogenesis. In addition, changes in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) gene expression were analysed at the level of rbcL protein and mRNA accumulation. AOX, PEPCase, and NAD-ME all increased only in clubs during development, increasing 5–6-fold by the latest prethermogenic stage and remained at this level. The induction of thermogenesis did not cause any changes in levels of AOX, indicating that SA does not affect levels of the enzyme itself, but instead must act to stimulate activity. Reported increases in NAD-ME activity during club development correlated closely with mRNA and protein accumulation, whereas PEPCase activity appears to be determined by post-transcriptional and post-translational processes. Interestingly, Rubisco protein and mRNA were found in relatively abundant amounts in clubs during early developmental stages, and disappeared rapidly as the thermogenic enzymes began to increase in abundance. The induction of thermogenesis by a synthetic inducer of plant pathogen resistance, 2,6-dichloroisonicotinic acid, as well as the involvement of SA and AOX in both processes, reinforces evidence for a link between mechanisms controlling disease resistance in all plants and thermogenic induction in Arum.

Salicylic acid (SA) induces resistance to all plant pathogens, including bacteria, fungi, and viruses, but the mechanism by which SA engenders resistance to viruses is not known. Pretreatment of tobacco mosaic virus (TMV)-susceptible (nn genotype) tobacco tissue with SA reduced the levels of viral RNAs and viral coat protein accumulating after inoculation with TMV. Viral RNAs were not affected equally, suggesting that SA treatment interferes with TMV replication. Salicylhydroxamic acid (SHAM), an inhibitor of the mitochondrial alternative oxidase, antagonized both SA-induced resistance to TMV in nn genotype plants and SA-induced acquired resistance in resistant (NN genotype) tobacco. SHAM did not inhibit induction of the PR-1 pathogenesis-related protein or induction of resistance to Erwinia carotovora or Botrytis cinerea by SA. This indicates that SA induces resistance to TMV via a novel SHAM-sensitive signal transduction pathway (potentially involving alternative oxidase), which is distinct from that leading to resistance to bacteria and fungi.