I. E. Moshkov

Nitric oxide in plants: an assessment of the current state of knowledge.

BACKGROUND AND AIMS After a series of seminal works during the last decade of the 20th century, nitric oxide (NO) is now firmly placed in the pantheon of plant signals. Nitric oxide acts in plant-microbe interactions, responses to abiotic stress, stomatal regulation and a range of developmental processes. By considering the recent advances in plant NO biology, this review will highlight certain key aspects that require further attention. SCOPE AND CONCLUSIONS The following questions will be considered. While cytosolic nitrate reductase is an important source of NO, the contributions of other mechanisms, including a poorly defined arginine oxidizing activity, need to be characterized at the molecular level. Other oxidative pathways utilizing polyamine and hydroxylamine also need further attention. Nitric oxide action is dependent on its concentration and spatial generation patterns. However, no single technology currently available is able to provide accurate in planta measurements of spatio-temporal patterns of NO production. It is also the case that pharmaceutical NO donors are used in studies, sometimes with little consideration of the kinetics of NO production. We here include in planta assessments of NO production from diethylamine nitric oxide, S-nitrosoglutathione and sodium nitroprusside following infiltration of tobacco leaves, which could aid workers in their experiments. Further, based on current data it is difficult to define a bespoke plant NO signalling pathway, but rather NO appears to act as a modifier of other signalling pathways. Thus, early reports that NO signalling involves cGMP-as in animal systems-require revisiting. Finally, as plants are exposed to NO from a number of external sources, investigations into the control of NO scavenging by such as non-symbiotic haemoglobins and other sinks for NO should feature more highly. By crystallizing these questions the authors encourage their resolution through the concerted efforts of the plant NO community.

Ethylene Regulates Monomeric GTP-Binding Protein Gene Expression and Activity in Arabidopsis

Ethylene rapidly and transiently up-regulates the activity of several monomeric GTP-binding proteins (monomeric G proteins) in leaves of Arabidopsis as determined by two-dimensional gel electrophoresis and autoradiographic analyses. The activation is suppressed by the receptor-directed inhibitor 1-methylcyclopropene. In theetr1-1 mutant, constitutive activity of all the monomeric G proteins activated by ethylene is down-regulated relative to wild type, and ethylene treatment has no effect on the levels of activity. Conversely, in the ctr1-1 mutant, several of the monomeric G proteins activated by ethylene are constitutively up-regulated. However, the activation profile of ctr1-1does not exactly mimic that of ethylene-treated wild type. Biochemical and molecular evidence suggested that some of these monomeric G proteins are of the Rab class. Expression of the genes for a number of monomeric G proteins in response to ethylene was investigated by reverse transcriptase-PCR. Rab8 andAra3 expression was increased within 10 min of ethylene treatment, although levels fell back significantly by 40 min. In theetr1-1 mutant, expression of Rab8 was lower than wild type and unaffected by ethylene; inctr1-1, expression of Rab8 was much higher than wild type and comparable with that seen in ethylene treatments. Expression in ctr1-1 was also unaffected by ethylene. Thus, the data indicate a role for monomeric G proteins in ethylene signal transduction.

The effect of ethylene and cytokinin on guanosine 5'-triphosphate binding and protein phosphorylation in leaves of Arabidopsis thaliana

Abstract. Binding of [α-32P]guanosine 5′-triphosphate ([α-32P]GTP) has been demonstrated in a Triton X-100-solubilised membrane fraction from leaves of Arabidopsis thaliana (L.) Heynh. Binding was stimulated by 1 h pre-treatment of leaves with ethylene and this effect was antagonised by the inclusion of N6-benzyladenine in the medium used for homogenisation. The ethylene-insensitive mutants eti5 and etr showed contrasting responses. In eti5 the constitutive level of GTP binding was higher than in the wild type whereas in etr the level was much lower. Neither ethylene nor cytokinin affected GTP binding in the mutants. The GTP-binding activity was localised in two bands at 22 and 25 kDa, both of which were immunoprecipitated by anti-pan-Ras antibodies, indicating that the activity is due to small GTP-binding proteins. In a similar membrane fraction, ethylene was shown to increase protein phosphorylation and benzyladenine antagonised this effect. In eti5 the constitutive level of protein phosphorylation was higher than in the wild type, but benzyladenine increased activity substantially while ethylene was without effect. In etr, protein phosphorylation was lower than in the wild type, ethylene was without effect, but cytokinin increased activity. A protein of Mr 17 kDa was detected on gels using antibodies to nucleoside diphosphate kinase. Phosphorylation of this protein was upregulated by ethylene but nucleoside diphosphate kinase activity was unaffected. The results are compared with the effect of the two hormones on the senescence of detached leaves and discussed in relation to pathways proposed for ethylene signal transduction.

Eukaryotic-like Ser/Thr protein kinases SpkC/F/K are involved in phosphorylation of GroES in the cyanobacterium Synechocystis

Serine/threonine protein kinases (STPKs) are the major participants in intracellular signal transduction in eukaryotes, such as yeasts, fungi, plants, and animals. Genome sequences indicate that these kinases are also present in prokaryotes, such as cyanobacteria. However, their roles in signal transduction in prokaryotes remain poorly understood. We have attempted to identify the roles of STPKs in response to heat stress in the prokaryotic cyanobacterium Synechocystis sp. PCC 6803, which has 12 genes for STPKs. Each gene was individually inactivated to generate a gene-knockout library of STPKs. We applied in vitro Ser/Thr protein phosphorylation and phosphoproteomics and identified the methionyl-tRNA synthetase, large subunit of RuBisCO, 6-phosphogluconate dehydrogenase, translation elongation factor Tu, heat-shock protein GrpE, and small chaperonin GroES as the putative targets for Ser/Thr phosphorylation. The expressed and purified GroES was used as an external substrate to screen the protein extracts of the individual mutants for their Ser/Thr kinase activities. The mutants that lack one of the three protein kinases, SpkC, SpkF, and SpkK, were unable to phosphorylate GroES in vitro, suggesting possible interactions between them towards their substrate. Complementation of the mutated SpkC, SpkF, and SpkK leads to the restoration of the ability of cells to phosphorylate the GroES. This suggests that these three STPKs are organized in a sequential order or a cascade and they work one after another to finally phosphorylate the GroES.

The effect of ethylene on GTP binding in extracts from pea epicotyls

Binding of [35S]guanosine 5′-o-(3-thiotriphosphate) (GTPγS) and of [α-32P]guanosine triphosphate ([α-32P]GTP) has been demonstrated in membrane preparations from epicotyl tips of etiolated plants ofPisum sativum L.; binding has also been shown in KCl-and Triton X-100-solubilised fractions from these membranes. Binding of GTPγS was of high affinity (KD = 3.17 × 10−8M), showed high specificity for guanine nucleotides and was stimulated by Mg2+ in the micromolar range. Binding was associated with only low levels of guanosine 5′-triphosphatase activity and was unaffected by treatment with mastoparan. In-vivo application of ethylene at 1 μl·l−1 stimulated GTP binding in fractions released from membranes by treatment with 750 mM KCl and Triton X-100. Affinity probing with ([α-32P]GTP) showed pronounced specific GTP binding to polypeptide(s) with relative molecular mass (Mr) of 28 kDa. The binding was stimulated markedly by ethylene and to some extent by AIF4−. Mouse monoclonal anti-pan-ras antibodies cross-reacted with several polypeptides in the 20 to 30-kDa region, and an [α-32P]GTP-labelled protein of Mr 28 kDa was precipitated by the same antibodies. The data indicate that the transduction of the ethylene signal may involve the intervention of GTP-binding proteins similar to the small monomeric GTP-binding proteins.

Ethylene rapidly up-regulates the activities of both monomeric GTP-binding proteins and protein kinase(s) in epicotyls of pea

It is demonstrated that, in etiolated pea (Pisum sativum) epicotyls, ethylene affects the activation of both monomeric GTP-binding proteins (monomeric G-proteins) and protein kinases. For monomeric G-proteins, the effect may be a rapid (2 min) and bimodal up-regulation, a transiently unimodal activation, or a transient down-regulation. Pretreatment with 1-methylcyclopropene abolishes the response to ethylene overall. Immunoprecipitation studies indicate that some of the monomeric G-proteins affected may be of the Rab class. Protein kinase activity is rapidly up-regulated by ethylene, the effect is inhibited by 1-methylcyclopropene, and the activation is bimodal. Immunoprecipitation indicates that the kinase(s) are of the MAP kinase ERK1 group. It is proposed that the data support the hypothesis that a transduction chain exists that is separate and antagonistic to that currently revealed by studies on Arabidopsis mutants.

Ethylene signal perception and transduction: multiple paradigms?

Current progress on the mechanisms of ethylene signal perception and transduction are reviewed with an emphasis on reconciling data from molecular genetics and from biochemical approaches. It is proposed that there exist two or more interacting transduction pathways.

Protein sensors and transducers of cold and osmotic stress in cyanobacteria and plants

Genome-wide analysis of gene expression at the transcriptional level with DNA microarrays identified almost all genes induced by particular stress in cyanobacteria and plants. Adaptation to stress conditions starts with the perception and transduction of the stress signal. A combination of systematic mutagenesis of potential sensors and transducers with genome transcription profiling allowed significant progress in understanding the mechanisms responsible for the perception of stress signals in photosynthesizing cells. The review considers the recent data on the cyanobacterial and plant signaling systems perceiving and transmitting the cold, hyperosmotic, and salt stress signals.

Interaction between ethylene and ABA in the regulation of polyamine level in Arabidopsis thaliana during UV-B stress

The effects of treatment with ethylene (0.01–100 μl/l) on ABA and polyamine contents and treatment with ABA on ethylene synthesis, polyamines content, and the resistance to UV-B radiation of two-week-old Arabidopsis thaliana (L.) Heynh, Columbia ecotype plants grown u⊋er sterile conditions were studied. Ethylene stimulated the accumulation of polyamines only at concentrations of 0.1–10 μl/l, which could activate ABA synthesis. Treatment with ABA (50–5000 μM, 1 μl per plant) decreased the UV-B-induced ethylene synthesis and a spermine and spermidine loss, increasing the content of putrescine, the precursor of these polyamines. ABA inhibited fresh weight accumulation in irradiated and nonirradiated plants but prevented them from severe damage and death at the high (18 kJ/m2) and lethal (27 kJ/m2) UV-B dose, respectively. The data obtained demonstrated a mutual regulation of ethylene and ABA syntheses and the participation of these hormones in the control of the polyamine level during adaptation of A. thaliana to UV-B stress.

Ethylene evolution and ABA and polyamine contents in Arabidopsis thaliana during UV-B stress

Changes in plant growth, membrane integrity, ethylene evolution, ABA content, and the content of free polyamines were examined in 14-day-old Arabidopsis thaliana (L.) Heynh., strain Columbia (Col-0) plants after a single UV-B irradiation with low (3 kJ/m2), moderate (6–9 kJ/m2), high (18 kJ/m2), and lethal (27 kJ/m2) doses. The UV-B treatment caused dose-dependent suppression of plant growth. One hour after irradiation, the membrane damage was evident from the increased leakage of electrolytes. The low-dose and moderate-dose irradiation caused a transient increase in evolution of ethylene and in the content of putrescine (spermidine and spermine precursor) with the peaks of these parameters attained at 5 and 24 h, respectively. The high-and lethaldose irradiation induced a smaller rise in ethylene evolution, with a slight trend to its decrease, especially, after the exposure to the lethal dose. The high and lethal doses of UV-B suppressed putrescine accumulation, depleted spermidine and spermine pools, and caused severe injuries and plant death. During the first day after irradiation, the ABA content increased in proportion to the irradiation dose. On the second day, the accumulation of ABA was observed in plants irradiated with moderate doses. The accumulation was arrested after a high-dose irradiation and was diminished by 45% after a lethal dose treatment. The results provide evidence for the involvement of ethylene, ABA, and polyamines in plant responses induced by UV-B irradiation.