Connections between sphingosine kinase and phospholipase D in the abscisic acid signaling pathway in Arabidopsis

Phosphatidic acid (PA) and phytosphingosine 1-phosphate (phyto-S1P) both are lipid messengers involved in plant response to abscisic acid (ABA). Our previous data indicate that PA binds to sphingosine kinase (SPHK) and increases its phyto-S1P-producing activity. To understand the cellular and physiological functions of the PA-SPHK interaction, we isolated Arabidopsis thaliana SPHK mutants sphk1-1 and sphk2-1 and characterized them, together with phospholipase Dα1 knock-out, pldα1, in plant response to ABA. Compared with wild-type (WT) plants, the SPHK mutants and pldα1 all displayed decreased sensitivity to ABA-promoted stomatal closure. Phyto-S1P promoted stomatal closure in sphk1-1 and sphk2-1, but not in pldα1, whereas PA promoted stomatal closure in sphk1-1, sphk2-1, and pldα1. The ABA activation of PLDα1 in leaves and protoplasts was attenuated in the SPHK mutants, and the ABA activation of SPHK was reduced in pldα1. In response to ABA, the accumulation of long-chain base phosphates was decreased in pldα1, whereas PA production was decreased in SPHK mutants, compared with WT. Collectively, these results indicate that SPHK and PLDα1 act together in ABA response and that SPHK and phyto-S1P act upstream of PLDα1 and PA in mediating the ABA response. PA is involved in the activation of SPHK, and activation of PLDα1 requires SPHK activity. The data suggest that SPHK/phyto-S1P and PLDα1A are co-dependent in amplification of response to ABA, mediating stomatal closure in Arabidopsis.     

Multiple actions of abscisic acid in senescence of oat leaves

The modifications induced by abscisic acid (ABA) on the senescence of oat leaves in darkness have been studied and are compared with its well-known effects in light. Contrary to the action in light, ABA preserves chlorophyll (Chl) in the dark almost as well as kinetin. Chlorophylla is decolorized more extensively thanb, and the content ofb is maintained by ABA almost at its initial level for 4 days. ABA also prevents proteolysis in darkness just as completely as chlorophyll loss, the relationship of both breakdown processes to ABA concentration being strictly log-linear over the range from 1 to 100 M. In line with this action, ABA inhibits formation of the neutral protease in the dark but not in the light. The data suggest that ABA and kinetin operate to preserve chlorophyll and protein by different mechanisms, since their actions are neither independent nor synergistic but actually interfere with one another. In this connection, protein values given by the Lowry and Bradford methods have been compared. In parallel with the effect on senescence, ABA slowly opens the stomata in the dark. This effect increases with time, and by day 3 the stomata in ABA are as open as in leaves on water in light. Thus all these effects of ABA in darkness are strikingly opposite to those commonly observed on leaves in natural lighting. In addition, ABA powerfully inhibits the formation of ethylene in the dark by the detached oat leaves, and this inhibition also tends to increase with time. Finally, a slight antagonism to ABA's action on senescence is exerted byp-coumaric acid in the light but not in the dark.     

Hydrogen peroxide is necessary for abscisic acid-induced senescence of rice leaves

The role of H₂O₂ in abscisic acid (ABA)-induced rice leaf senescence is investigated. ABA treatment resulted in H₂O₂ production in rice leaves, which preceded the occurrence of leaf senescence. Dimethylthiourea, a chemical trap for H₂O₂, was observed to be effective in inhibiting ABA-induced senescence, ABA-increased malondialdehyde (MDA) content, ABA-increased antioxidative enzyme activities (superoxide dismutase, ascorbate peroxidase, glutathione reductase and catalase), and ABA-decreased antioxidant contents (ascorbic acid and reduced glutathione) in rice leaves. Diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibitors of NADPH oxidase, and KCN and NaN₃, inhibitors of peroxidase, prevented ABA-induced H₂O₂ production, suggesting NADPH oxidase and peroxidase are H₂O₂-generating enzymes in ABA-treated rice leaves. DPI, IMD, KCN, and NaN₃ also inhibited ABA-promoted senescence, ABA-increased MDA contents, ABA-increased antioxidative enzyme activities, and ABA-decreased antioxidants in rice leaves. These results suggest that H₂O₂ is involved in ABA-induced senescence of rice leaves.     

Nitric oxide counteracts the senescence of rice leaves induced by abscisic acid

In the present study, we evaluate the protective effect of nitric oxide (NO) against senescence of rice leaves promoted by ABA. Senescence of rice leaves was determined by the decrease of protein content. ABA treatment resulted in (1) induction of leaf senescence, (2) increase in H2O2 and malondialdehyde (MDA) contents, (3) decrease in reduced form glutathione (GSH) and ascorbic acid (AsA) contents, and (4) increase in antioxidative enzyme activities (superoxide dismutase, ascorbate peroxidase, glutathione reductase, and catalase). All these ABA effects were reduced by free radical scavengers such as sodium benzoate and GSH. NO donors [N-tert-butyl-alpha-phenylnitrone (PBN), sodium nitroprusside, 3-morpholinosydonimine, and AsA + NaNO2] were effective in reducing ABA-induced leaf senescence. PBN prevented ABA-induced increase in the contents of H2O2 and MDA, decrease in the contents of GSH and AsA, and increase in the activities of antioxidative enzymes. The protective effect of PBN on ABA-promoted senescence, ABA-increased H2O2 content and lipid peroxidation, ABA-decreased GSH and AsA, and ABA-increased antioxidative enzyme activities was reversed by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a NO-specific scavenger, suggesting that the protective effect of PBN is attributable to NO released. Reduction of ABA-induced senescence by NO in rice leaves is most likely mediated through its ability to scavenge active oxygen species including H2O2.     

Plasmalemma abscisic acid perception leads to RAB18 expression via phospholipase D activation in Arabidopsis suspension cells

Abscisic acid (ABA) plays a key role in the control of stomatal aperture by regulating ion channel activities and water exchanges across the plasma membrane of guard cells. Changes in cytoplasmic calcium content and activation of anion and outward-rectifying K(+) channels are among the earliest cellular responses to ABA in guard cells. In Arabidopsis suspension cells, we have demonstrated that outer plasmalemma perception of ABA triggered similar early events. Furthermore, a Ca(2+) influx and the activation of anion channels are part of the ABA-signaling pathway leading to the specific expression of RAB18. Here, we determine whether phospholipases are involved in ABA-induced RAB18 expression. Phospholipase C is not implicated in this ABA pathway. Using a transphosphatidylation reaction, we show that ABA plasmalemma perception results in a transient stimulation of phospholipase D (PLD) activity, which is necessary for RAB18 expression. Further experiments showed that PLD activation was unlikely to be regulated by heterotrimeric G proteins. We also observed that ABA-dependent stimulation of PLD was necessary for the activation of plasma anion current. However, when ABA activation of plasma anion channels was inhibited, the ABA-dependent activation of PLD was unchanged. Thus, we conclude that in Arabidopsis suspension cells, ABA stimulation of PLD acts upstream from anion channels in the transduction pathway leading to RAB18 expression.     

Endogenous gibberellin and abscisic Acid content as related to senescence of detached lettuce leaves

Levels of gibberillins (GAs) and of abscisic acid (ABA) in attached leaves of romaine lettuce (Lactuca sativa L.) declined as the leaf became older. The time course of changes in hormone levels, determined in detached lettuce leaves kept in darkness, revealed that a sharp decline in GAs accompanied by a moderate rise in ABA occurred before the onset of chlorophyll degradation. As senescence advanced, no GAs could be detected and a considerable rise of ABA was observed. A similar sequence of hormonal modifications, but more pronounced, was observed in the course of accelerated senescence induced by either Ethephon or water stress. When kinetin or GA₃ was applied to detached leaves, the loss of chlorophyll and the rise in ABA were reduced. Bound GAs were detected in senescent leaves. They were not found in the kinetin-treated leaves, which contained a relatively high level of free GAs. The results suggest that senescence in detached romaine lettuce leaves is connected with a depletion of free GAs and cytokinins, which is thereafter followed by a great surge in ABA.     

Genomics approach to abscisic acid- and gibberellin-responsive genes in rice.

We used an 8987-EST collection to construct a cDNA microarray system with various genomics information (full-length cDNA, expression profile, high accuracy genome sequence, phenotype, genetic map, and physical map) in rice. This array was used as a probe to hybridize target RNAs prepared from normally grown callus of rice and from callus treated for 6 hr or 3 days with the hormones abscisic acid (ABA) or gibberellin (GA). We identified 509 clones, including many clones that had never been annotated as ABA-or GA-responsive. These genes included not only ABA- or GA-responsive genes but also genes responsive to other physiological conditions such as pathogen infection, heat shock, and metal ion stress. Comparison of ABA- and GA-responsive genes revealed antagonistic regulation for these genes by both hormones except for one defense-related gene, thionin. The gene for thionin was up-regulated by both hormone treatments for 3 days. The upstream regions of all the genes that were regulated by both hormones had cis-elements for ABA and GA response. We performed a clustering analysis of genes regulated by both hormones and various expression profiles that showed three notable clusters (seed tissues, low temperature and sugar starvation, and thionin-gene related). A comparison of the cis-elements for hormone response genes between rice and Arabidopsis thaliana, we identified cis-elements for dehydration-stress response or for expression of amylase gene as Arabidopsis gene-specific or rice gene-specific, respectively.     

Antisense and chemical suppression of the nonmevalonate pathway affects ent-kaurene biosynthesis in Arabidopsis

Transgenic plants of Arabidopsis thaliana (L.) Heynh. (ecotype Columbia) expressing the antisense AtMECT gene, encoding 2- C-methyl- D-erythritol 4-phosphate cytidylyltransferase, were generated to elucidate the physiological role of the nonmevalonate pathway for production of ent-kaurene, the latter being the plastidic precursor of gibberellins. In transformed plants pigmentation and accumulation of ent-kaurene were reduced compared to wild-type plants. Fosmidomycin, an inhibitor of 1-deoxy- D-xylulose 5-phosphate reductoisomerase (DXR), caused a similar depletion of these compounds in transgenic plants. These observations suggest that both AtMECT and DXR are important in the synthesis of isopentenyl diphosphate and dimethylallyl diphosphate and that ent-kaurene is mainly produced through the nonmevalonate pathway in the plastid.     

Inhibition of phospholipase D alpha by N-acylethanolamines

N-Acylethanolamines (NAEs) are endogenous lipids in plants produced from the phospholipid precursor, N-acylphosphatidylethanolamine, by phospholipase D (PLD). Here, we show that seven types of plant NAEs differing in acyl chain length and degree of unsaturation were potent inhibitors of the well-characterized, plant-specific isoform of PLD-PLD alpha. It is notable that PLD alpha, unlike other PLD isoforms, has been shown not to catalyze the formation of NAEs from N-acylphosphatidylethanolamine. In general, inhibition of PLD alpha activity by NAEs increased with decreasing acyl chain length and decreasing degree of unsaturation, such that N-lauroylethanolamine and N-myristoylethanolamine were most potent with IC(50)s at submicromolar concentrations for the recombinant castor bean (Ricinus communis) PLD alpha expressed in Escherichia coli and for partially purified cabbage (Brassica oleracea) PLD alpha. NAEs did not inhibit PLD from Streptomyces chromofuscus, and exhibited only moderate, mixed effects for two other recombinant plant PLD isoforms. Consistent with the inhibitory biochemical effects on PLD alpha in vitro, N-lauroylethanolamine, but not lauric acid, selectively inhibited abscisic acid-induced closure of stomata in epidermal peels of tobacco (Nicotiana tabacum cv Xanthi) and Commelina communis at low micromolar concentrations. Together, these results provide a new class of biochemical inhibitors to assist in the evaluation of PLD alpha physiological function(s), and they suggest a novel, lipid mediator role for endogenously produced NAEs in plant cells.     

Role of growth regulators in the senescence of Arabidopsis thaliana leaves

A homozygous, dominant, C₂H₄-resistant line of Arabidopsis thaliana (L.) Heynh (cv. Columbia; er ) was selected from ethylmethylsulfonate-mutagenized seed, and used to test the role of C₂H₄ and other growth regulators in senescence of mature leaves. Chlorophyll (Chl) loss from disks excised from leaves of er was much slower than that from wild-type (WT) disks, whether they were held in the light or in the dark. C₂H₄ accelerated Che loss from WT disks but had no effect on the yellowing of mutant disks. C₂H₄ biosynthesis was higher in disks from the mutant plants, particularly in the light. In the dark, treatment with the cytokinin, 6-benzyladenine (BA), reduced Chl loss from wild-type disks, but had no effect on mutant disks. In the light, BA treatment stimulated chlorophyll breakdown in both wild type and mutant disks. Treatment with abscisic acid (ABA) stimulated chlorophyll loss in wild-type and mutant disks, whether they were held in the light or the dark. C₂H₄ production was stimulated in ABA-treated disks, but they still yellowed even when C₂H₄ production was inhibited by application of aminooxyacetic acid (AOA). These data indicate that C₂H₄ is only one of the factors involved in leaf senescence, and that the promotion of senescence by ABA is not mediated through its stimulation of C₂H₄ production.     

Characterization of microsomal and mitochondrial phospholipase D activities and cloning of a phospholipase D alpha cDNA from strawberry fruits.

Phospholipase D alpha (PLD, EC 3.1.4.4)) is a key enzyme involved in membrane deterioration that occurs during fruit ripening and senescence. The biochemical and molecular characteristics of PLD was studied in strawberry (Fragaria ananassa Duch) fruits, which are non-climacteric fruits. PLD activity was primarily associated with the mitochondrial and microsomal fractions and showed increased activity during development. Optimal pH levels of activity were observed at 5.5 and 6.5 for mitochondrial PLD and at 5 and 7 for microsomal PLD. Calcium enhanced microsomal PLD activity at 1-40 microM levels. PLD activity followed Michaelis-Menten kinetics. Lineweaver-Burk analysis gave Km values in the range of 114 and 277 microM using dipalmitoylphosphatidylcholine (DPPC) as substrate for mitochondrial and microsomal PLD, respectively. The Vmax value for the microsomal PLD was nearly 12-fold higher than that of mitochondrial PLD. A 2874 bp full-length cDNA for PLD alpha was amplified from strawberry fruit mRNA using RT-PCR and 5'- and 3'-RACE encoding an 810 amino acid-polypeptide. The predicted strawberry PLD sequence showed the characteristic C2 domain and the phospholipase domains conferring calcium sensitivity and the enzyme activity, respectively. The strawberry PLD alpha showed a high degree of similarity to other PLD alphas from plants. The implications of PLD regulation during ripening of fruits are discussed.     

Profiling membrane lipids in plant stress responses. Role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis

A sensitive approach based on electrospray ionization tandem mass spectrometry has been employed to profile membrane lipid molecular species in Arabidopsis undergoing cold and freezing stresses. Freezing at a sublethal temperature induced a decline in many molecular species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) but induced an increase in phosphatidic acid (PA) and lysophospholipids. To probe the metabolic steps generating these changes, lipids of Arabidopsis deficient in the most abundant phospholipase D, PLD alpha, were analyzed. The PC content dropped only half as much, and PA levels rose only half as high in the PLD alpha-deficient plants as in wild-type plants. In contrast, neither PE nor PG levels decreased significantly more in wild-type plants than in PLD alpha-deficient plants. These data suggest that PC, rather than PE and PG, is the major in vivo substrate of PLD alpha. The action of PLD alpha during freezing is of special interest because Arabidopsis plants that are deficient in PLD alpha have improved tolerance to freezing. The greater loss of PC and increase in PA in wild-type plants as compared with PLD alpha-deficient plants may be responsible for destabilizing membrane bilayer structure, resulting in a greater propensity toward membrane fusion and cell death in wild-type plants.     

Involvement of plasma-membrane NADPH oxidase in abscisic acid- and water stress-induced antioxidant defense in leaves of maize seedlings

The roles of the plasma-membrane (PM) NADPH oxidase in abscisic acid (ABA)- and water stress-induced antioxidant defense were investigated in leaves of maize ( Zea mays L.) seedlings. Treatment by exogenous ABA (100 micro M ABA) or osmotic stress (-0.7 MPa induced by polyethylene glycol) significantly increased the activity of the PM NADPH oxidase, the production of leaf O(2)(-), the activities of several antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase), and the contents of antioxidant metabolites (ascorbate and reduced glutathione). Pretreatment with three different inhibitors of NADPH oxidase (diphenylene iodonium, imidazole and pyridine) or an inhibitor of ABA biosynthesis (tungstate) reduced the increase in the activity of the PM NADPH oxidase and the production of leaf O(2)(-), and the capacity of antioxidant defense systems mediated by ABA. The inhibitory effects above caused by tungstate were reversed by exogenous ABA. These data indicate that NADPH oxidase is involved in the ABA-induced production of active oxygen species (AOS), and our results depict a minimal chain of events initiated by water stress-induced ABA accumulation, which then triggers the production of AOS by membrane-bound NADPH oxidase, resulting in the induction of antioxidant defense systems against oxidative damage in plants.     

The interplay between cytokinins and light during senescence in detached Arabidopsis leaves

Light and cytokinins are known to be the key players in the regulation of plant senescence. In detached leaves, the retarding effect of light on senescence is well described; however, it is not clear to what extent is this effect connected with changes in endogenous cytokinin levels. We have performed a detailed analysis of changes in endogenous content of 29 cytokinin forms in detached leaves of Arabidopsis thaliana (wild‐type and 3 cytokinin receptor double mutants). Leaves were kept under different light conditions, and changes in cytokinin content were correlated with changes in chlorophyll content, efficiency of photosystem II photochemistry, and lipid peroxidation. In leaves kept in darkness, we have observed decreased content of the most abundant cytokinin free bases and ribosides, but the content of cis‐zeatin increased, which indicates the role of this cytokinin in the maintenance of basal leaf viability. Our findings underscore the importance of light conditions on the content of specific cytokinins, especially N⁶‐(Δ²‐isopentenyl)adenine. On the basis of our results, we present a scheme summarizing the contribution of the main active forms of cytokinins, cytokinin receptors, and light to senescence regulation. We conclude that light can compensate the disrupted cytokinin signalling in detached leaves.     

The different fates of mitochondria and chloroplasts during dark-induced senescence in Arabidopsis leaves

Senescence is an active process allowing the reallocation of valuable nutrients from the senescing organ towards storage and/or growing tissues. Using Arabidopsis thaliana leaves from both whole darkened plants (DPs) and individually darkened leaves (IDLs), we investigated the fate of mitochondria and chloroplasts during dark-induced leaf senescence. Combining in vivo visualization of fates of the two organelles by three-dimensional reconstructions of abaxial parts of leaves with functional measurements of photosynthesis and respiration, we showed that the two experimental systems displayed major differences during 6 d of dark treatment. In whole DPs, organelles were largely retained in both epidermal and mesophyll cells. However, while the photosynthetic capacity was maintained, the capacity of mitochondrial respiration decreased. In contrast, IDLs showed a rapid decline in photosynthetic capacity while maintaining a high capacity for mitochondrial respiration throughout the treatment. In addition, we noticed an unequal degradation of organelles in the different cell types of the senescing leaf. From these data, we suggest that metabolism in leaves of the whole DPs enters a 'stand-by mode' to preserve the photosynthetic machinery for as long as possible. However, in IDLs, mitochondria actively provide energy and carbon skeletons for the degradation of cell constituents, facilitating the retrieval of nutrients. Finally, the heterogeneity of the degradation processes involved during senescence is discussed with regard to the fate of mitochondria and chloroplasts in the different cell types.     

Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana

The location of tryptophan residues in the actin macromolecule was studied on the basis of the known 3D structure. For every tryptophan residue the polarity and packing density of their microenvironments were evaluated. To estimate the accessibility of the tryptophan residues to the solvent molecules it was proposed to analyze the radial dependence of the packing density of atoms in the macromolecule about the geometric center of the indole rings of the tryptophan residues. The proposed analysis revealed that the microenvironment of tryptophan residues Trp-340 and Trp-356 has a very high density. So these residues can be regarded as internal and inaccessible to solvent molecules. Their microenvironment is mainly formed by non-polar groups of protein. Though the packing density of the Trp-86 microenvironment is lower, this tryptophan residue is apparently also inaccessible to solvent molecules, as it is located in the inner region of macromolecule. Tryptophan residue Trp-79 is external and accessible to the solvent. All residues that can affect tryptophan fluorescence were revealed. It was found that in the close vicinity of tryptophan residues Trp-79 and Trp-86 there are a number of sulfur atoms of cysteine and methionine residues that are known to be effective quenchers of tryptophan fluorescence. The most essential is the location of SG atom of Cys-10 near the NE1 atom of the indole ring of tryptophan residue Trp-86. On the basis of microenvironment analysis of these tryptophan residues and the evaluation of energy transfer between them it was concluded that the contribution of tryptophan residues Trp-79 and Trp-86 must be low. Intrinsic fluorescence of actin must be mainly determined by two other tryptophan residues--Trp-340 and Trp-356. It is possible that the unstrained conformation of tryptophan residue Trp-340 and the existence of aromatic rings of tyrosine and phenylalanine and proline residues in the microenvironments of tryptophan residues Trp-340 and Trp-356 are also essential to their blue fluorescence spectrum.     

Copper amine oxidase and phospholipase D act independently in abscisic acid (ABA)-induced stomatal closure in Vicia faba and Arabidopsis

Recent evidence has demonstrated that both copper amine oxidase (CuAO; EC 1.4.3.6) and phospholipase D (PLD; EC 3.1.4.4) are involved in abscisic acid (ABA)-induced stomatal closure. In this study, we investigated the interaction between CuAO and PLD in the ABA response. Pretreatment with either CuAO or PLD inhibitors alone or that with both additively led to impairment of ABA-induced H₂O₂ production and stomatal closure in Vicia faba. ABA-stimulated PLD activation could not be inhibited by the CuAO inhibitor, and CuAO activity was not affected by the PLD inhibitor. These data suggest that CuAO and PLD act independently in the ABA response. To further examine PLD and CuAO activities in ABA responses, we used the Arabidopsis mutants cuaoζ and pldα1. Ablation of guard cell-expressed CuAOζ or PLDα1 gene retarded ABA-induced H₂O₂ generation and stomatal closure. As a product of PLD, phosphatidic acid (PA) substantially enhanced H₂O₂ production and stomatal closure in wide type, pldα1, and cuaoζ. Moreover, putrescine (Put), a substrate of CuAO as well as an activator of PLD, induced H₂O₂ production and stomatal closure in WT but not in both mutants. These results suggest that CuAO and PLD act independently in ABA-induced stomatal closure.