Apyrases, extracellular ATP and the regulation of growth

Although no definitive receptor for extracellular ATP (eATP) has been identified in plants, there is now stronger physiological evidence that the effects of eATP on plant growth are mediated by a receptor, or, as in animals, by multiple receptors. Recent papers clarify how extracellular nucleotides induce changes in [Ca(2+)](cyt), and the production of nitric oxide (NO) and reactive oxygen species. They document links between eATP signaling and the synthesis or transport of hormones, and they reveal that applied nucleotides can regulate the aperture of stomates, which release ATP when stimulated by light and hormones. Ectoapyrases (ecto-nucleoside triphosphate-diphosphohydrolase) help control both the diverse signaling changes and downstream growth changes induced by extracellular nucleotides by limiting their concentration in the extracellular matrix (ECM).     

植物细胞外信号分子eATP研究进展

细胞外ATP(extracellular ATP, eATP)是目前公认的细胞外信号分子, 参与调控多种环境刺激下植物的生长、发育和防御反应。在植物细胞信号转导过程中, eATP具有双重功能, 其作用主要取决于细胞外基质中eATP的浓度。eATP含量过高或者过低都会导致细胞死亡, 适度水平的eATP则有助于植物的生长和发育。细胞外三磷酸腺苷双磷酸酶(Apyrase)严格控制细胞外基质中eATP的水平, 因此有助于调控植物在逆境条件下的生长和防御反应。该文总结了植物中eATP的发现、产生和清除以及受体和信号转导等研究进展, 重点论述eATP在逆境条件下的生理功能, 并对植物eATP的研究方向作了展望。     

Extracellular ATP signaling and homeostasis in plant cells

Extracellular ATP (eATP) is now recognized as an important signaling agent in plant growth and defense response to environmental stimuli. eATP has dual functions in plant cell signaling, which is largely dependent on its concentration in the extracellular matrix (ECM). A lethal level of eATP (extremely low or high) causes cell death, whereas a moderate level of eATP benefits plant growth and development. Ecto-apyrases (Nucleoside Triphosphate-Diphosphohydrolase) help control the eATP concentrations in the ECM, and thus contributing to the mediation of plant growth and defense response upon environmental stress. In this review, we summarize eATP signaling in plants and highlight the correlation between eATP homeostasis control and programmed cell death.     

Extracellular ATP: a potential regulator of plant cell death

Adenosine 5′‐triphosphate (ATP) has been regarded as an intracellular energy currency molecule for many years. In recent decades, it has been determined that ATP is released into the extracellular milieu by animal, plant and microbial cells. In animal cells, this extracellular ATP (eATP) functions as a signalling compound to mediate many cellular processes through its interaction with membrane‐associated receptor proteins. It has also been reported that eATP is a signalling molecule required for the regulation of plant growth, development and responses to environmental stimuli. Recently, the first plant receptor for eATP was identified in Arabidopsis thaliana. Interestingly, some studies have shown that eATP is of particular importance in the control of plant cell death. In this review article, we summarize and discuss the theoretical and experimental advances that have been made with regard to the roles and mechanisms of eATP in plant cell death. We also make an attempt to address some speculative aspects to help develop and expand future research in this area.     

Extracellular ATP and nitric oxide signaling pathways regulate redox-dependent responses associated to root hair growth in etiolated Arabidopsis seedlings

Extracellular ATP (eATP) and nitric oxide (NO) have emerged as crucial players in plant development, stress responses and cell viability. Glutathione (GSH) is an abundant reducing agent with proposed roles in plant growth, development and stress physiology. In a recent publication, we demonstrated that eATP and NO restore hypocotyl elongation of etiolated Arabidopsis seedlings treated with GSH. Here it is reported that exogenous ATP also restores root hair growth suggesting a role for ATP and NO in the regulation of redox balance associated to specific processes of plant morphogenesis. A tentative model integrating redox-, eATP- and NO-signaling pathways during root hair growth in Arabidopsis seedlings is presented.Key words: Arabidopsis thaliana, extracellular ATP signaling, nitric oxide, redox system, root hair     

Extracellular ATP in plants. Visualization, localization, and analysis of physiological significance in growth and signaling

Extracellular ATP (eATP) in animals is well documented and known to play an important role in cellular signaling (e.g. at the nerve synapse). The existence of eATP has been postulated in plants; however, there is no definitive experimental evidence for its presence or an explanation as to how such a polar molecule could exit the plant cell and what physiological role it may play in plant growth and development. The presence of eATP in plants (Medicago truncatula) was detected by constructing a novel reporter; i.e. fusing a cellulose-binding domain peptide to the ATP-requiring enzyme luciferase. Application of this reporter to plant roots allowed visualization of eATP in the presence of the substrate luciferin. Luciferase activity could be detected in the interstitial spaces between plant epidermal cells and predominantly at the regions of actively growing cells. The levels of eATP were closely correlated with regions of active growth and cell expansion. Pharmacological compounds known to alter cytoplasmic calcium levels revealed that ATP release is a calcium-dependent process and may occur through vesicular fusion, an important step in the polar growth of actively growing root hairs. Reactive oxygen species (ROS) activity at the root hair tip is not only essential for root hair growth, but also dependent on the cytoplasmic calcium levels. Whereas application of exogenous ATP and a chitin mixture increased ROS activity in root hairs, no changes were observed in response to adenosine, AMP, ADP, and nonhydrolyzable ATP (betagammameATP). However, application of exogenous potato (Solanum tuberosum) apyrase (ATPase) decreased ROS activity, suggesting that cytoplasmic calcium gradients and ROS activity are closely associated with eATP release.     

Plant responses to extracellular nucleotides: Cellular processes and biological effects

Higher plants exhibit cellular responsiveness to the exogenous application of purine nucleotides in a manner consistent with a cell–cell signaling function for these molecules. Like animals, plants respond to extracellular ATP, ADP, and stable analogues (e.g., ATPγS and ADPβS) by increasing the cytoplasmic concentration of calcium. Agonist substrate specificity and concentration dependency suggest receptor mediation of these events, and, although the identity of the plant receptor is currently unknown, pharmacological analysis points to the involvement of a plasma membrane-localized calcium channel. Extracellular ATP can also induce the production of reactive oxygen species and stimulate an increase in the mRNA levels of a number of stress- and calcium-regulated genes, suggesting a role for nucleotide-based signaling in plant wound and defense responses. Furthermore, the growth and development of plants can also be altered by the application of external ATP. Recent studies are only beginning to uncover the complexities of plant signaling networks activated in response to extracellular ATP and how these might interact to affect plant physiological processes.     

Homeostasis of extracellular ATP in uninfected RBCs from a Plasmodium falciparum culture and derived microparticles

Plasmodium falciparum, a dangerous parasitic agent causing malaria, invades human red blood cells (RBCs), causing hemolysis and microvascular obstruction. These and other pathological processes of malaria patients are due to metabolic and structural changes occurring in uninfected RBCs. In addition, infection activates the production of microparticles (MPs).ATP and byproducts are important extracellular ligands modulating purinergic signaling within the intravascular space.Here, we analyzed the contribution of uninfected RBCs and MPs to the regulation of extracellular ATP (eATP) of RBCs, which depends on the balance between ATP release by specific transporters and eATP hydrolysis by ectonucleotidases.RBCs were cultured with P. falciparum for 24–48 h prior to experiments, from which uninfected RBCs and MPs were purified. On-line luminometry was used to quantify the kinetics of ATP release. Luminometry, colorimetry and radioactive methods were used to assess the rate of eATP hydrolysis by ectonucleotidases. Rates of ATP release and eATP hydrolysis were also evaluated in MPs.Uninfected RBCs challenged by different stimuli displayed a strong and transient activation of ATP release, together with an elevated rate of eATP hydrolysis. MPs contained ATP in their lumen, which was released upon vesicle rupture, and were able to hydrolyze eATP.Results suggest that uninfected RBCs and MPs can act as important determinants of eATP regulation of RBCs during malaria.The comparison of eATP homeostasis in infected RBCs, ui-RBCs, and MPs allowed us to speculate on the impact of P. falciparum infection on intravascular purinergic signaling and the control of the vascular caliber by RBCs.     

Extracellular ATP is a potent signaling molecule in the activation of the Japanese flounder (Paralichthys olivaceus) innate immune responses

Innate immunity is the first line of defense against pathogen infections. Extracellular ATP (eATP) is one of the most studied danger-associated molecular pattern molecules that can activate host innate immune responses through binding with and activating purinergic receptors on the plasma membrane. The detailed actions of eATP on fish innate immunity, however, remain poorly understood. In this study, we investigated bacterial pathogen-induced ATP release in head kidney cells of the Japanese flounder Paralichthys olivaceus. We also examined the actions of eATP on pro-inflammatory cytokine and immune-related gene expression, the activity of induced NO synthase (iNOS), and the production of reactive oxygen species (ROS) and NO in Japanese flounder immune cells. We demonstrate that ATP is dynamically released from Japanese flounder head kidney cells into the extracellular milieu during immune challenge by formalin-inactivated Edwardsiella tarda and Vibrio anguillarum. In addition, we show that eATP administration results in profound up-regulation of pro-inflammatory cytokine gene expression, iNOS activity, and inflammatory mediator production, including ROS and NO, in Japanese flounder immune cells. Altogether, our findings demonstrate that eATP is a potent signaling molecule for the activation of innate immune responses in fish.     

非生物胁迫对烟草悬浮细胞胞内和胞外ATP水平的影响

以烟草悬浮细胞BY-2(Nicotiana tabacum ‘Bright Yellow-2’)为材料,研究了NaCl、PEG(6000)、低温3种非生物胁迫对细胞内ATP(intracellular ATP,iATP)和细胞外ATP(extracellular ATP,eATP)水平的影响。结果显示：50～200 mmol·L^-1 NaCl处理导致烟草悬浮细胞膜通透性显著增加(P<0.05),100和200 mmol·L^-1 NaCl处理时iATP和eATP水平显著降低(P<0.05)。随着PEG质量浓度的增加(50、100、200 g·L^-1),烟草悬浮细胞膜通透性和eATP水平逐渐增加,其中在200 g·L^-1 PEG处理时eATP水平显著增加至对照的3.4倍(P<0.05),而iATP水平则在200 g·L^-1 PEG处理时显著降低至对照的0.5倍(P<0.05)。0～10℃低温处理后,烟草悬浮细胞膜通透性和iATP水平呈不同程度增加,其中0℃处...     

Proteomic Analysis of Extracellular ATP-Regulated Proteins Identifies ATP Synthase β-Subunit as a Novel Plant Cell Death Regulator

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.ATP is a ubiquitous, energy-rich molecule of fundamental importance in living organisms. It is a key substrate and vital cofactor in many biochemical reactions and is thus conserved by all cells. However, in addition to its localization and functions inside cells, ATP is actively secreted to the extracellular matrix where it forms a halo around the external cell surface. The existence of this extracellular ATP (eATP)¹ has been reported in several organisms including bacteria (1), primitive eukaryotes (2), animals (3), and plants (4–6). This eATP is not wasted, but harnessed at the cell surface as a potent signaling molecule enabling cells to communicate with their neighbors and regulate crucial growth and developmental processes.In animals, eATP is a crucial signal molecule in several physiological processes such as neurotransmission (7, 8), regulation of blood pressure (9), enhanced production of reactive oxygen species (ROS) (10), protein translocation (11), and apoptosis (12). Extracellular ATP signal perception at the animal cell surface is mediated by P2X and P2Y receptors, which bind ATP extracellularly and recruit intracellular second messengers (13, 14). P2X receptors are ligand-gated ion channels that provide extracellular Ca²⁺ a corridor for cell entry after binding eATP, facilitating a surge in cytosolic [Ca²⁺] that is essential in activating down-stream signaling. P2Y receptors transduce the eATP signal by marshalling heteromeric G-proteins on the cytosolic face of the plasma membrane and activating appropriate downstream effectors.Although eATP exists in plants, homologous P2X/P2Y receptors for eATP signal perception have not yet been identified, even in plant species with fully sequenced genomes. Notwithstanding the obscurity of plant eATP signal sensors, some of the key downstream messengers recruited by eATP-mediated signaling are known. For example, eATP triggers a surge in cytosolic Ca²⁺ concentration (15–17) and a heightened production of nitric oxide (18–20) and reactive oxygen species (17, 21, 22). Altering eATP levels is attended by activation of plant gene expression (16, 21) and changes in protein abundance (5, 23), indicating that eATP-mediated signaling impacts on plant physiology. Indeed eATP has been demonstrated to regulate plant growth (20, 24–26), gravitropic responses (27), xenobiotic resistance (4), plant-symbiont interactions (28), and plant-pathogen interactions (23, 29). However, the mechanism by which eATP regulates these processes remains unclear, largely because the eATP signal sensors and downstream signal regulatory genes and proteins have not been identified.We previously reported that eATP plays a central regulatory role in plant cell death processes (5). Therefore, an understanding of the signaling components galvanized by eATP in cell death regulation might serve a useful purpose in providing mechanistic detail of how eATP signals in plant physiological processes. We found that eATP-mediated signaling negatively regulates cell death as its removal by application of ATP-degrading enzymes to the apoplast activates plant cell death (5). Remarkably, fumonisin B1 (FB1), a pathogen-derived molecule that activates defense gene expression in Arabidopsis (30), commandeers this eATP-regulated signaling to trigger programmed cell death (5). FB1 is a mycotoxin secreted by fungi in the genus Fusarium and initiates programmed cell death in both animal and plant cells (31, 32). In Arabidopsis, FB1 inaugurates cell death by inactivating eATP-mediated signaling via triggering a drastic collapse in the levels of eATP (5). FB1-induced Arabidopsis programmed cell death is dependent on the plant signaling hormone salicylic acid (33), which is a key regulator of eATP levels (29). Because concurrent application of FB1 and exogenous ATP to remedy the FB1-induced eATP deficit blocks death, FB1 and exogenous ATP treatments can therefore be used as probes to identify the key signal regulators downstream of eATP in cell death control. This is vital for achieving the global objective of elucidating the mechanism of eATP signaling in plant physiology.Gel-based proteomic analyses have been previously applied to successfully identify the novel role of eATP in the regulation of plant defense gene expression and disease resistance (23, 29). We have now employed FB1 and ATP treatments together with two-dimensional difference in-gel electrophoresis (DIGE) and matrix-assisted laser desorption-time of flight MS (MALDI-TOF MS) to identify the changes in Arabidopsis protein profiles associated with a shift from normal to cell death-inception metabolism. Additional reverse genetic analyses enabled us to definitively identify a putative ATP synthase β-subunit as a target for eATP-mediated signaling with an unexpected function in the regulation of plant programmed cell death.     

Extracellular ATP-induced NO production and its dependence on membrane Ca2+ flux in Salvia miltiorrhiza hairy roots

Extracellular ATP (eATP) is a novel signalling agent, and nitric oxide (NO) is a well-established signal molecule with diverse functions in plant growth and development. This study characterizes NO production induced by exogenous ATP and examines its relationship with other important signalling agents, Ca(2+) and H(2)O(2) in Salvia miltiorrhiza hairy root culture. Exogenous ATP was applied at 10-500 microM to the hairy root cultures and stimulated NO production was detectable within 30 min. The NO level increased with ATP dose from 10-100 microM but decreased from 100-200 muM or higher. The ATP-induced NO production was mimicked by a non-hydrolysable ATP analogue ATPgammaS, but only weakly by ADP, AMP or adenosine. The ATP-induced NO production was blocked by Ca(2+) antagonists, but not affected by a protein kinase inhibitor. ATP also induced H(2)O(2) production, which was dependent on both Ca(2+) and protein kinases, and also on NO biosynthesis. On the other hand, ATP induced a rapid increase in the intracellular Ca(2+) level, which was dependent on NO but not H(2)O(2). The results suggest that NO is implicated in ATP-induced responses and signal transduction in plant cells, and ATP signalling is closely related to Ca(2+) and ROS signalling.     

胞外ATP对壳聚糖诱导的ROS和PAL活性变化的影响

以烟草BY-2悬浮细胞为材料,探讨了胞外ATP对壳聚糖引起的活性氧(reactive oxygen species,ROS)水平和苯丙氨酸解氨酶(phenylalanine ammonia-lyase,PAL)活性变化的影响。结果表明,5~20μg·mL^-1壳聚糖处理导致了烟草悬浮细胞细胞内ROS水平逐渐增加;壳聚糖也导致了PAL活性的增加,其活性在15μg·mL^-1壳聚糖处理下达到峰值,此后有所降低。10~40μmol·L^-1外源ATP处理未引起烟草悬浮细胞内ROS水平和PAL活性的显著变化。细胞外ATP水平则随壳聚糖浓度的增加而逐渐下降。本文进一步分析了细胞外ATP对壳聚糖引起的ROS水平和PAL活性变化的影响。结果显示,外源施加20μmol·L^-1ATP可以有效降低壳聚糖诱导的烟草悬浮细胞ROS水平上升,同时外源ATP也明显减缓了壳聚糖所诱导的PAL活性的上升。上述结果表明,细胞外ATP水平能够影响壳聚糖引起的ROS水平和PAL活性的变化。     

The signaling role of extracellular ATP and its dependence on Ca2+ flux in elicitation of Salvia miltiorrhiza hairy root cultures

The application of a polysaccharide elicitor from yeast extract,YE, to Salvia miltiorrhiza hairy root cultures induced transientrelease of ATP from the roots to the medium, leading to a dose-dependentincrease in the extracellular ATP (eATP) level. The eATP levelrose to a peak (about 6.5 nM with 100 mg l^–1 YE) at about10 h after YE treatment, but dropped to the control level 6h later. The elicitor-induced ATP release was dependent on membraneCa²⁺ influx, and abolished by the Ca²⁺ chelator EGTA or thechannel blocker La³⁺. The YE-induced H₂O₂ production was stronglyinhibited by reactive blue (RB), a specific inhibitor of membranepurinoceptors. On the other hand, the application of exogenousATP at 10–100 µM to the cultures also induced rapidand dose-dependent increases in H₂O₂ production and medium pH,both of which were effectively blocked by RB and EGTA. The non-hydrolyzableATP analog ATPS was as effective as ATP, but the hydrolyzedderivatives ADP or AMP were not so effective in inducing thepH and H₂O₂ increases. Our results suggest that ATP releaseis an early event and that eATP plays a signaling role in theelicitation of plant cell responses; Ca²⁺ is required for activationof the elicitor-induced ATP release and the eATP signal transduction.This is the first report on ATP release induced by a fungalelicitor and its involvement in the elicitor-induced responsesin plant cells.     

不同光强下细胞外三磷酸腺苷对菜豆叶片光化学反应特性的影响

三磷酸腺苷(ATP)不但分布在细胞内部, 而且广泛存在于动物和植物细胞的细胞外基质中。细胞外ATP (eATP)可与细胞膜表面相应的受体结合并激发细胞内的第二信使, 从而调节细胞的多种生理学功能。但目前对于eATP是否也能对植物的光合作用产生影响则研究较少。该文以菜豆(Phaseolus vulgaris)叶片为实验材料, 研究了在不同光强下eATP对菜豆叶片叶绿素荧光特性和光合放氧速率的影响。结果显示, 随着光强的增加, 叶片的光适应下最大光化学效率(F_v′/F_m′)、光系统II (PSII)实际光化学效率(Y(II))、光化学猝灭系数(q_P)均呈现下降趋势, 而电子传递速率(ETR)、非光化学猝灭系数(q_N)以及调节性能量耗散的量子产量(Y(NPQ))随着光强的增加呈上升趋势。与对照相比, eATP的处理可以显著提高菜豆叶片PSII的潜在最大光化学效率(F_v/F_m)、Y(II)、q_P、ETR和光合放氧速率; 但eATP的处理对F_v′/F_m′、q_N以及Y(NPQ)没有显著影响。AMP-PCP (β,γ-亚甲基三磷酸腺苷, eATP细胞外受体的抑制剂)的处理显著降低了F_v/F_m、F_v′/F_m′、Y(II)、ETR和光合放氧速率, 同时也显著增加了q_N以及Y(NPQ)的水平。以上结果显示, 植物eATP水平的变化对植物光合作用的光化学反应有着重要的影响。     

Extracellular ATP functions as an endogenous external metabolite regulating plant cell viability

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.     

Extracellular ATP induces the accumulation of superoxide via NADPH oxidases in Arabidopsis

Extracellular ATP can serve as a signaling agent in animal cells, and, as suggested by recent reports, may also do so in plant cells. In animal cells it induces the production of reactive oxygen species through the mediation of NADPH oxidase. Similarly, here we report that in leaves of Arabidopsis (Arabidopsis thaliana), applied ATP, but not AMP or phosphate, induces the accumulation of superoxide (O2-) in a biphasic, dose-dependent manner, with a threshold at 500 nm ATP. This effect did not require ATP hydrolysis for it was mimicked by ATPgammaS. ATP also induced increased levels of Arabidopsis respiratory burst oxidase homolog D (AtrbohD) mRNA, but ATP-treated plants that had disrupted AtrbohD and AtrbohF genes did not accumulate O2-, indicating that NADPH oxidases are responsible for the induced O2- accumulation. Inhibitors of mammalian P2-type ATP receptors abolished ATP-induced O2- production, suggesting that the ATP effects may be mediated through P2-like receptors in plants. Cytosolic Ca2+ and calmodulin are likely to help transduce the ATP responses, as they do in animal cells, because a Ca2+ channel blocker, a Ca2+ chelator, and calmodulin antagonist all reduced ATP-induced O2- accumulation. Furthermore, ATP treatment enhanced the expression of genes that are induced by wounds and other stresses. The ATP measured at wound sites averaged 40 microm, well above the level needed to induce O2- accumulation and gene expression changes. Transgenic plants overexpressing an apyrase gene had reduced O2- production in response to applied ATP and wounding. Together, these data suggest a possible role for extracellular ATP as a signal potentially in wound and stress responses.     

Phototropin signaling and stomatal opening as a model case

Phototropins are plant-specific light-activated receptor kinases that regulate diverse blue-light-induced responses, and serve to optimize plant growth under various light environments. Phototropins undergo autophosphorylation as an essential step for their signaling and induce a variety of tissue-specific or organ-specific responses, but the divergent mechanisms for these responses are unknown. It is most likely that the phototropins generate a specific output after the event of autophosphorylation. In this report, we will review the common steps of phototropin signaling and the numerous interactive proteins of phototropins, which may act as signal transducers for the diverse responses. We also describe the phototropin-mediated signaling process of stomatal guard cells and its crosstalk with abscisic acid signaling.     

Proteomic analysis of extracellular ATP-regulated proteins identifies ATP synthase beta-subunit as a novel plant cell death regulator

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.