Günther F. E. Scherer

Polyamines, polyamine oxidases and nitric oxide in development, abiotic and biotic stresses

Nitric oxide (NO), polyamines (PAs), diamine oxidases (DAO) and polyamine oxidases (PAO) play important roles in wide spectrum of physiological processes such as germination, root development, flowering and senescence and in defence responses against abiotic and biotic stress conditions. This functional overlapping suggests interaction of NO and PA in signalling cascades. Exogenous application of PAs putrescine, spermidine and spermine to Arabidopsis seedlings induced NO production as observed by fluorimetry and fluorescence microscopy using the NO-binding fluorophores DAF-2 and DAR-4M. The observed NO release induced by 1 mM spermine treatment in the Arabidopsis seedlings was very rapid without apparent lag phase. These observations pave a new insight into PA-mediated signalling and NO as a potential mediator of PA actions. When comparing the functions of NO and PA in plant development and abiotic and biotic stresses common to both signalling components it can be speculated that NO may be a link between PA-mediated stress responses filing a gap between many known physiological effects of PAs and amelioration of stresses. NO production indicated by PAs could be mediated either by H(2)O(2), one reaction product of oxidation of PAs by DAO and PAO, or by unknown mechanisms involving PAs, DAO and PAO.

Rapid increase of NO release in plant cell cultures induced by cytokinin.

4,5‐Diaminofluorescein, a fluorescence indicator for NO, was applied to detect the release of NO from plant cells. NO production was increased within 3 min when plant cell cultures (Arabidopsis, parsley, and tobacco) were treated by cytokinin and was dose‐dependent and signal‐specific in that other plant hormones and inactive cytokinin analog were not effective in stimulating of NO release. The response was quenched by addition of 2‐(aminoethyl)‐2‐thiopseudourea, an inhibitor of the animal NO synthase, and by addition of an NO scavenger, 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐1‐oxy‐3‐oxide. These results imply that NO may act in cytokinin signal transduction.

Molecular Identification of Cytosolic, Patatin-Related Phospholipases A from Arabidopsis with Potential Functions in Plant Signal Transduction

Rapid activation of phospholipase A (PLA) by auxin or plant-pathogen interaction suggests a function in signal transduction for this enzyme, but the molecular identification of a cytosolic PLA carrying out this function remains open. We isolated four cDNA sequences from Arabidopsis (ecotype Columbia), AtPLA I,AtPLA IIA, AtPLA IVA, andAtPLA IVC, which are members of the patatin-related PLA gene family in plants and which are homologous to the animal Ca2+-independent PLA2 gene family. Expression was measured by reverse transcriptase-polymerase chain reaction, andAtPLA I transcripts were found preferentially in shoots,AtPLA IIA and AtPLA IVA in roots, andAtPLA IVC in flowers. Transient expression of the four PLA-green fluorescent protein fusion proteins in tobacco (Nicotiana tabacum) leaves showed they were located in the cytosol and not in the vacuoles. Surprisingly,AtPLA::green fluorescent protein was also localized to chloroplasts. The enzymatic activity of the purified recombinant AtPLA IVA toward phosphatidylcholine was dependent on Ca2+, saturated at 0.5 mm, and had a pH optimum of about 7.0. It had both PLA1 and PLA2 specificity. The enzyme showed in vitro highest sensitivity toward the PLA2 inhibitors palmitoyltrifluoromethyl ketone (PACOCF3,K i approximately 30 nm), arachidonyltrifluoromethyl ketone (AACOCF3,K i approximately 25 μm), and tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one (K i approximately 200 nm) and was also sensitive to other previously used inhibitors 5,8,11,14-eicosatetraynoic acid (K iapproximately 3 μm) and nordihydroguajaretic acid (K i approximately 15 μm). The influence of these PLA2 inhibitors on elongation in etiolated Arabidopsis seedlings was tested, and tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one and 5,8,11,14-eicosatetraynoic acid inhibited hypocotyl elongation maximally at concentrations close to theirK i in vitro.

Patatin-related phospholipase A: nomenclature, subfamilies and functions in plants

The release of fatty acids from membrane glycerolipids has been implicated in a variety of cellular processes, but the enzymes involved and their regulation are poorly understood in plants. One large group of acyl-hydrolyzing enzymes is structurally related to patatins. Patatins are potato tuber proteins with acyl-hydrolyzing activity, and the patatin catalytic domain is widely spread in bacterial, yeast, plant and animal enzymes. Recent results have indicated that patatin-related enzymes are involved in different cellular functions, including plant responses to auxin, elicitors or pathogens, and abiotic stresses and lipid mobilization during seed germination. In this review, we highlight recent developments regarding these enzymes and propose the nomenclature pPLA for the patatin-related phospholipase A enzyme.

COPPER AMINE OXIDASE1 (CuAO1) of Arabidopsis thaliana Contributes to Abscisic Acid-and Polyamine-Induced Nitric Oxide Biosynthesis and Abscisic Acid Signal Transduction

Polyamines (PA), polyamine oxidases, copper amine oxidases, and nitric oxide (NO) play important roles in physiology and stress responses in plants. NO biosynthesis as a result of catabolism of PA by polyamine oxidases and copper amine oxidases may explain in part PA-mediated responses. Involvement of a copper amine oxidase gene, COPPER AMINE OXIDASE1 (CuAO1), of Arabidopsis was tested for its role in stress responses using the knockouts cuao1-1 and cuao1-2. PA-induced and ABA-induced NO production investigated by fluorometry and fluorescence microscopy showed that the cuao1-1 and cuao1-2 are impaired in NO production, suggesting a function of CuAO1 in PA and ABA-mediated NO production. Furthermore, we found a PA-dependent increase in protein S-nitrosylation. The addition of PA and ABA also resulted in H(2)O(2) increases. cuao1-1 and cuao1-2 showed less sensitivity to exogenous ABA supplementation during germination, seedling establishment, and root growth inhibition as compared to wild-type. In response to ABA treatment, expression levels of the stress-responsive genes RD29A and ADH1 were significantly lower in the knockouts. These observations characterize cuao1-1 and cuao1-2 as ABA-insensitive mutants. Taken together, our findings extend the ABA signal transduction network to include CuAO1 as one potential contributor to enhanced NO production by ABA.

The heterozygous abp1/ABP1 insertional mutant has defects in functions requiring polar auxin transport and in regulation of early auxin‐regulated genes

AUXIN-BINDING PROTEIN 1 (ABP1) is not easily accessible for molecular studies because the homozygous T-DNA insertion mutant is embryo-lethal. We found that the heterozygous abp1/ABP1 insertion mutant has defects in auxin physiology-related responses: higher root slanting angles, longer hypocotyls, agravitropic roots and hypocotyls, aphototropic hypocotyls, and decreased apical dominance. Heterozygous plants flowered earlier than wild-type plants under short-day conditions. The length of the main root, the lateral root density and the hypocotyl length were little altered in the mutant in response to auxin. Compared to wild-type plants, transcription of early auxin-regulated genes (IAA2, IAA11, IAA13, IAA14, IAA19, IAA20, SAUR9, SAUR15, SAUR23, GH3.5 and ABP1) was less strongly up-regulated in the mutant by 0.1, 1 and 10 μm IAA. Surprisingly, ABP1 was itself an early auxin-up-regulated gene. IAA uptake into the mutant seedlings during auxin treatments was indistinguishable from wild-type. Basipetal auxin transport in young roots was slower in the mutant, indicating a PIN2/EIR1 defect, while acropetal transport was indistinguishable from wild-type. In the eir1 background, three of the early auxin-regulated genes tested (IAA2, IAA13 and ABP1) were more strongly induced by 1 μm IAA in comparison to wild-type, but eight of them were less up-regulated in comparison to wild-type. Similar but not identical disturbances in regulation of early auxin-regulated genes indicate tight functional linkage of ABP1 and auxin transport regulation. We hypothesize that ABP1 is involved in the regulation of polar auxin transport, and thus affects local auxin concentration and early auxin gene regulation. In turn, ABP1 itself is under the transcriptional control of auxin.

Roles of Arabidopsis Patatin-Related Phospholipases A in Root Development Are Related to Auxin Responses and Phosphate Deficiency

Phospholipase A enzymes cleave phospho- and galactolipids to generate free fatty acids and lysolipids that function in animal and plant hormone signaling. Here, we describe three Arabidopsis patatin-related phospholipase A (pPLA) genes AtPLAIVA, AtPLAIVB, and AtPLAIVC and their corresponding proteins. Loss-of-function mutants reveal roles for these pPLAs in roots during normal development and under phosphate deprivation. AtPLAIVA is expressed strongly and exclusively in roots and AtplaIVA-null mutants have reduced lateral root development, characteristic of an impaired auxin response. By contrast, AtPLAIVB is expressed weakly in roots, cotyledons, and leaves but is transcriptionally induced by auxin, although AtplaIVB mutants develop normally. AtPLAIVC is expressed in the floral gynaecium and is induced by abscisic acid (ABA) or phosphate deficiency in roots. While an AtplaIVC-1 loss-of-function mutant displays ABA responsiveness, it exhibits an impaired response to phosphate deficiency during root development. Recombinant AtPLA proteins hydrolyze preferentially galactolipids and, less efficiently, phospholipids, although these enzymes are not localized in chloroplasts. We find that AtPLAIVA and AtPLAIVB are phosphorylated by calcium-dependent protein kinases in vitro and this enhances their activities on phosphatidylcholine but not on phosphatidylglycerol. Taken together, the data reveal novel functions of pPLAs in root development with individual roles at the interface between phosphate deficiency and auxin signaling.

Down-regulation by elicitors of phosphatidylcholine-hydrolyzing phospholipase C and up-regulation of phospholipase A in plant cells

Phosphatidylcholine, labeled by two fluorescent fatty acids, was fed to cultured plant cells (Petrosilenum crispum, L.; VBI-0, Nicotiana benthiana, L.) and fluorescent diacylglycerol (DAG) was the major metabolite. When a glycoprotein elicitor, derived from Phytophthora sojae, was applied to the parsley cells and the small protein cryptogein from Phytophthora cryptogea was applied to the tobacco cells, these signal substances strongly and rapidly decreased the pool of fluorescent diacylglycerol and weakly increased the pool of free fluorescent fatty acid and of fluorescent lysophosphatidylcholine. The cells responded in a very similar way to the application of mastoparan, a wasp venom peptide. As phosphatidic acid was only a very minor fluorescent metabolite DAG is hypothesized to arise by the action of a phosphatidylcholine-hydrolyzing phospholipase C which was down-regulated by elicitors. Up-regulation of a phospholipase A by elicitors is also suggested by these results. This is the first evidence for phosphatidylcholine-hydrolyzing phospholipase C in plant signal transduction.

Inhibitors of animal phospholipase A2 enzymes are selective inhibitors of auxin-dependent growth. Implications for auxin-induced signal transduction

Abstract. Auxin and elicitors reportedly activate phospolipase A. A number of inhibitors known to inhibit animal phospholipase A2 were tested for their ability to inhibit hormone and fusicoccin-induced growth. To this end, growth induced by indolyl-3-acetic acid and 2,4-dichlorophenoxyacetic acid in hypocotyl segments of etiolated zucchini (Cucurbita pepo L.) seedlings was determined in the presence of the inhibitors nordihydroguajaretic acid (NDGA), aristolochic acid, 5,8,11,14-eicosatetraynoic acid (ETYA), PBx (a prostaglandin derivative), and oleylethyl phosphocholine. Each chemical proved inhibitory to auxin-induced growth, oleylethyl phosphocholine being the least effective. The effects of the first three inhibitors were investigated in more detail. Growth induced by 10 μM 2,4-dichlorophenoxyacetic acid or 1 μM indolyl-3-acetic acid was inhibited 50% by about 30–50 μM NDGA, by about 25 μM aristolochic acid, and by about 10–20 μM EYTA. Growth inhibition was reversible and became apparent 0.5–1 h after inhibitor addition. Growth induced by 0.5 or 1 μM fusicoccin was much less inhibited by NDGA and by ETYA, whereas aristolochic acid was only slightly less effective on fusicoccin-induced than on auxin-induced growth. These three inhibitors were also tested for their effects on gibberellin-induced growth in light-grown peas (Pisum sativum L.) and on cytokinin-induced expansion growth in excised cotyledons from radish (Raphanus sativum L.) seedlings. In both tests, aristolochic acid had toxic side-effects although gibberellin-induced growth was still apparent. In the gibberellin test, neither NDGA at up to 100 μM nor ETYA at 80 μM was inhibitory to hormone-induced growth. Moreover, 40 μM ETYA was not inhibitory to kinetin-induced growth. We hypothesize that the selectivity of phospholipase A2 inhibitors for auxin-induced growth implies a different signal transduction pathway for each of the different signal substances tested, and that auxins might use fatty acid(s) and/or lysophospholipid(s) or their derivatives as the preferred second messengers.

NO donors mimic and NO inhibitors inhibit cytokinin action in betalaine accumulation in Amaranthus caudatus.

A classical biotest for cytokinin is based on the accumulation of betalainesin Amaranthus species. We have shown that inhibitorsof nitric oxide synthase from animals inhibit this response and that chemicaldonors of nitric oxide (NO) stimulate betalaine biosynthesis in Amaranthuscaudatus. NO could be an intermediate in cytokinin signalling.