Ya'acov Leshem

Evidence for the function of the free radical gas - nitric oxide (NO°)-as an endogenous maturation and senescence regulating factor in higher plants

Abstract The hypothesis that endogenously-produced nitric oxide (NO • ) gas is a natural plant growth regulator was experimentally tested. Experimentation encompassed a wide spectrum of both climacteric and non-climacteric varieties of fruits, flowers, vegetables and legume sprout species. Specific NO • probe monitoring of both vegetative and generative plant organs revealed that temporal progress of maturation and senescence goes hand in hand with a significant decrease of NO • emission. Conversely, exogenous application of the gas either by direct fumigation in an O 2 -free atmosphere or by means of NO • releasing biochemicals such as N- tert -butyl-α-phenylnitrone and 3 morpholinosyl-nonimine, markedly delayed senescence and maturation. NO • decrease was moreover stoichiometrically related to ethylene upsurge, this phenomenon also being geared to ethylene increment under short-term heat stress. Furthermore, a classic foliage expansion bioassay indicated a concentration-dependent growth effect evidenced as a typical growth regulator-like response where low concentrations promote, and high concentrations inhibit growth. Regarded together, experimental data lends support to the possible role of NO • as a natural senescence-delaying plant growth regulating agent acting primarily, but not solely, by down regulating ethylene emission.

The Characterization and Contrasting Effects of the Nitric Oxide Free Radical in Vegetative Stress and Senescence of Pisum sativum Linn. Foliage

Summary As monitored respectively by a nitric oxide (NO)-specific probe and gas chromatography, emission of both NO and ethylene from pea foliage increases with duration of stress and/or senescence promoting conditions. Depending on concentration, NO appears to exert a stress-coping or, contrariwise, an inhibitory effect on leaf growth. Stress coping is evidenced by the marked deceleration of stress ethylene by application of the NO releasing compound S-nitroso-N-acetylpenicillamine (SNAP) and possibly also by significant increment of leaf disc expansion upon treatment with low concentrations of chemically generated NO gas. At higher NO concentrations, leaf expansion is inhibited by NO treatment as well as by three different NO releasing compounds: SNAP, 3-morpholinosydnonimine, and N-tert-butyl-α-phenyl-nitrone. Further evidence to the assumption of endogenous involvement of NO metabolism is lent by the inhibition reversal induced by the nitric oxide synthase inhibitor NG-methyl-L-arginine. Reconciliation of the opposite effects is discussed in terms of translocation and cellular binding sites of the free radical gas.

Plant senescence processes and free radicals

Free radicals acting at sensitive subcellular sites, appear to play a pivotal role in both the deleterious and beneficial effects of maturation and senescence of various plant organs--leaves, flowers, and fruit. As evidenced by ESR spectrometry, spin trapping, specific membrane phase transition studies and enzyme kinetics, an important factor in the above processes appears to be lipoxygenase activity producing polyunsaturated fatty acid (PUFA) hydroperoxides and subsequently several free radical species and senescence-promoting compounds such as ethylene, malondialdehyde and jasmonic acid. The most intensely investigated are the oxy-free radical species including O2-., .OH, RO., ROO., PUFA and semiquinone free radicals. Higher plants are equipped with ways and means to combat free radicals and these may be classified under two general headings; (a) direct scavengers including SOD, ascorbic acid, and alpha-tocopherol acting in concert (b) incipient preventative mechanisms against radical formation, these include xanthine oxidase inhibitors, strategies based on endogenous H2O2 disposal in the form of peroxidative enzymes and glutathione turnover, and Ca2+ channel blockers. The antisenescence phytohormone cytokinin appears to possess a dual effect and may act in both capacities. The special case of delayed free radical formation in comparatively dry biological systems such as seeds is detailed, and specific free radical-generating photosensitizer compounds are also discussed.

Nitric oxide in biological systems

Against a background of a general description of the action of the free radical gas nitric oxide (NO), some of its close derivatives such as peroxynitrite, and of its formative enzyme nitric oxide synthase (NOS) in mammals, possible plant analogies are discussed. Resembling endogenous effects in mammals which may be either promotory or inhibitory, a possible physiological if not anatomical parallel pertains to NOs vasodilatory effects. The latter in mammals inter alia are encountered in extensible coronary-, blood vessel lining-, endotheliar-, pulmonary-and penile tissue while in plants such effects may be on cell wall matrix components which constrain potential turgor-promoted cell expansion. The same final outcome in both plants and mammals may be enhancement of periplasmic membrane fluidity. As with certain aspects of NO action in mammals, the nature of the response may be concentration-dependent — lower ones, promotory — higher, inhibitory. As deduced from trials on pea foliage, an apparent linkage of NO and ethylene metabolism is specific to plants. Thus, low concentrations of either endogenously-produced or exogenously-applied NO in the 10−6 M range exert significant growth-promoting and ethylene-inhibiting effects which are reversed by higher NO concentrations or by equimolar applications of the NOS inhibitor-NG-methyl-L-arginine or of NO releasing compounds. An overview is given of other modes of NO action in biological systems and of basic issues of NO and NOS function in plant growth regulation which await elucidation.

FUMIGATION WITH NITRIC OXIDE TO EXTEND THE POSTHARVEST LIFE OF STRAWBERRIES

Abstract Strawberry (Fragaria ananassa Duch. cv. Pajaro) fruit were fumigated with nitric oxide immediately after harvest. Fumigation was performed in an anaerobic nitrogen atmosphere for up to 2 h at 20°C at nitric oxide concentrations from 1.0 to 4000 μl l−1 then held at 20 and 5°C in air containing 0.1 μl l−1 ethylene, a concentration prevalent in ambient air at fruit and vegetable markets. Treatment at both temperatures extended the postharvest life of strawberries, the most pronounced effect being obtained with nitric oxide in the concentration range 5–10 μl l−1 which produced >50% extension in shelf life. The possibility of commercial application is discussed.

Harnessing Senescence Delaying Gases Nitric Oxide and Nitrous Oxide: A Novel Approach to Postharvest Control of Fresh Horticultural Produce

The effects of ethylene on postharvest horticultural produce are a major limiting factor in their orderly and efficient marketing. Nitric oxide and nitrous oxide have recently been shown to inhibit ethylene action and synthesis in higher plants and their potential for commercial use to extend the storage and marketing life of fruits, vegetables and flowers is reviewed.

Nitric oxide degradation in oxygen atmospheres and rate of uptake by horticultural produce

Abstract Fumigation of horticultural produce for a few hours with low concentrations of nitric oxide (NO) has been previously shown to extend postharvest life, but treatments have been carried out in an atmosphere with very low oxygen (O 2 ) due to the known rapid oxidation of NO. This study examined the rate of loss of 30 μl l −1 NO from atmospheres containing 0.3–21% O 2 and found that the rate of loss was much lower than expected with the half life of NO ranging from about 16 h in 0.3% O 2 to 3.5 h in 21% O 2 . Exposure of ten types of produce to 30–100 μl l −1 NO showed that the rate of loss of NO was greater at higher concentrations and was much greater than in air only, the difference indicating uptake of NO by produce. The rate of uptake of NO by produce varied considerably, with a 130-fold range from about 90 mmol min −1 per 100 g in lettuces to about 1 mmol min −1 per 100 g in lime. When calculated on a surface area basis, the range was only 15-fold varying between about 1 and 13 mmol min −1 per 100 cm 2 , but with no obvious pattern that could be related to produce characteristics. NO thus may be sufficiently stable at the concentrations and fumigation times required for produce to be treated in normal air, although surface area and other as yet unknown physical characteristics of produce need to be taken into account as these affect the quantity of gas absorbed.

Biophysical parameters of linoleic acid hydroperoxides as assessed by surface behavior and Fourier Transform Infra-Red spectrometry: possible pertinence to senescence

Hydroperoxidation of linoleic acid (LA) by two major lipoxygenases (LOX) produced octadecanoic trans, cis (Δ 10, 12) 9-hydroperoxide (9 LAHP) and octadecanoic cis, trans (Δ, 9, 11) 13-hydroperoxide (13, LAHP). The effect of hydroperoxidation on surface tension-related parameters of simulated monolayer membranes was studied. Langmuir-Blodgett isotherms demonstrated that hydroperoxidation of LA markedly reduces molecular area, and hence increases monolayer rigidity. The reduction in the surface area of 13 LAHP was more pronounced than that of 9 LAHP. Surface tension and Contact Angle measurements demonstrated a similar effect. However, in contrast to the molecular area data, both HP species behaved similarly in that as compared to the unoxidized LA, they both increased Contact angles to the same extent. Fourier-Transform Infra-Red studies indicated that 13 LAHP undergoes non-enzymatic cyclization, and hence molecular area reduction. The results described are discussed in terms of membrane senescence and possible relevance to plant prostaglandin-like compounds of the jasmonate group.

Biophysical Compaction Studies of Effects of Abscisic Acid on Membrane Phospholipids: Possible Role in Stomatal Physiology and Senescence

Summary In vitro biopysical compaction studies based on Langmuir tensiometry and Contact Angle measurements combined with Mass Spectral in vivo characterization of dipalmitoylphosphatidylcholine (DPPC) in guard cell microsomal membranes has indicated that abscisic acid (ABA) may exert a specifically located compacting effect. It is suggested that the rigidity imposed by the ABA-sensitive DPPC, in contrast to other membranes, as in lung surfactant tissue may fulfill a collapse-resisting function in stomatal physiology.

Inhibition of oxidative catabolism and oxy free radical production as a possible mode of cytokinin control of foliar senescence

Increment of levels of both lipoxygenase and superoxide-dismutase which typically increase with age or upon light deprivation is significantly lowered by cytokinin treatment. It is suggested that cytokinin on the one hand by means of inhibition of polyunsaturated fatty acid catabolism prevents incipient formation of free radicals while on the other serves as a scavenger of radicals already formed.