BOTANY!!! (de Juan, Gamboa, Manalaysay, Matundan, Sebastian)

Detoxification of Dissolved SO2 (Bisulfite) by Terricolous Mosses

Group 1De Juan, Michelle Ligaya E.

Gamboa, Domina Flor L.Manalaysay, Jessica Alba G.Matundan, Celine Marie C.

*Sulfur dioxide exists in these forms: SO2, SO3-2

and HSO3- (bisulfite)

*Terricolous: Land-dwelling

INTRODUCTION

Pleurozium schreberi- calcifuge moss- moderately tolerant of SO2

Rhytidiadelphus triquetrus

- calcareous or calcicole moss- strongly affected by SO2 pollution in the 20th century

Pleurozium schreberi

Source: http://commons.wikimedia.org/wiki/File:Rhytidiadelphus_triquetrus.JPG

Rhytidiadelphus triquetrus

Source: http://commons.wikimedia.org/wiki/File:Pleurozium.schreberi.jpg

⇨ Tolerance of SO2 is seen on the metabolic

detoxification of dissolved bisulfite by these mosses

Bryophytes (as well as Lichens) are sensitive to atmospheric pollution, particularly SO2

- Limited cuticle- High surface area- Low metabolic activities- Modest innate growth rates

* Detoxification ⇨ Resistant bryophytes: relatively high growth rates: which means that they have the ability to detoxify

* In higher vascular plants: tolerance on SO2 is by detoxification mechanisms (excluding tolerance by cuticle and stomata)

oxidized to sulfate ion (SO4-) ORreduced to sulfide

*SO2 Phytotoxicity

Intracellular O2-

production as cause of SO2 phytotoxicity

*Photo-oxidation: oxidation in the presence of radiant energy (light)

*SO2 Phytotoxicity

Intracellular O2-

production as cause of SO2 phytotoxicity

Superoxide dismutase- Active in SO2 tolerant plants

- Catalyses decomposition of O2-

- Inhibits photo-oxidation of SO2

*Photo-oxidation: oxidation in the presence of radiant energy (light)

*SO2 Phytotoxicity

Intracellular O2-

production as cause of SO2 phytotoxicity

Superoxide dismutase- Active in SO2 tolerant plants

- Catalyses decomposition of O2-

- Inhibits photo-oxidation of SO2

Diethyl dithiocarbamate (DETC)- Controls activity of superoxide

dismutase

*Photo-oxidation: oxidation in the presence of radiant energy (light)

* Detoxification like higher vascular plants in bryophytes: Sphagnum- Higher tolerance for plants in more polluted areas- Oxidation of bisulfite: brought about by metal cations (Fe3+, Mn2+ and Cu2+)

*Focus of the studyPleurozium schreberiRhytidiadelphus triquetrus

⇨ In dilute bisulfite solutions: Photosynthesis in these mosses was strongly inhibited by short (2-hour) incubations with bisulfite ⇨ Longer incubations: no effect → Shoots have a high capacity to detoxify dissolved SO2

*Hypotheses(a) tolerance of bisulfite by P. schreberi and R.

triquetrus depends primarily on detoxification (oxidation) of the pollutant

(b) bisulfite detoxification involves metabolic energy

(c) Ca2+ and Fe3+ stimulate the detoxification process

(d) SOD is involved in bisulfite detoxification

MATERIALS AND METHODS

(1) Pleurozium schreberi: collected from an acid, sandy loam soil under grassland and scrub

(2) Rhytidiaelphus triquetrus: was collected from chalk grassland on a rendzina soil

(1) Incubation treatmentsShort-term incubation experimentLong-term incubation experiment

* Reagent used: NaHSO3

(2) Bisulfite disappearance in relation to initial concentration

(3) DCMU experiment (3-( 30,40-dichlorophenyl)-1,1-dimethylurea)- Inhibits photosynthetic electron transport and oxygen evolution

(4) DETC experiment (diethyldithiocarbamate)- Inhibitor of superoxide dismutase- To see if the enzyme plays a role in bisulfite oxidation

(5) Bisulfite oxidation: influence of Ca2+ and Fe3+

(6) Bisulfite and sulfate determinations- Spectrophotometric methods

(7) Statistical analyses- One-way ANOVA- Duncan’s multiple range test

RESULTS

* Disappearance of bisulfite during short-term incubations with moss shoots

- Decay of bisulfite greater in the presence of light for both species

- Rhytidiadelphus triquetrus: has a greater capacity in catalyzing disappearance of bisulfite

*Disappearance of bisulfite during long-term incubations

- Absence of mosses: 28% remaining at the end of the fifth day of incubation

- Presence of mosses: ≈ 95% of bisulfite had disappeared after a three day incubation

*Disappearance of bisulfite in relation to initial concentration

- Disappearance of bisulfite: dependent on initial concentration and presence of light

(1) Pleurozium schreberi: initial concentration of bisulfite is roughly proportional to the final; no significant effect of light

(2) Rhytidiadelphus triquetrus: total bisulfite lost increased in the presence of light

*Disappearance of bisulfite in relation to acidity

- pH 3 to 5: small reduction in final volume of bisulfite for both species* Much greater reduction at pH 6

*Effects of DCMU on disappearance of bisulfite

- Presence of DCMU in both light and dark: great bisulfite persistence

- Twice as much bisulfite remained in the incubation solutions of Pleurozium schreberi as in those of Rhytidiadelphus triquetrus

*Effects of DETC on disappearance of bisulfite

- inhibited bisulfite loss from the incubation medium in both species

- Pleurozium schreberi: doubling of the concentration of bisulfite remaining

- Rhytidiadelphus triquetrus: even greater increase in concentration of bisulfite remaining

*Evidence for extracellular oxidation of bisulfite to sulfate: influence of Ca and Fe

- Pleurozium schreberiFe: no differenceCa: more sulfate was detectedEDTA: no change or small increase in sulfate

- Rhytidiadelphus triquetrusFe: small but significant increase in sulfateCa: reduction in sulfate productionEDTA: reduction in sulfate

DISCUSSION

- Addition of bisulfiteRapid cessation of photosynthesisIncreasing incubation periods: photosynthesis restored→ Due to oxidation of bisulfite

- Presence of the two mossesGreatly accelerates decrease in amount of bisulfite in incubation solutions

- Rate of bisulfite loss depends on:(1) Presence or absence of light(2) Application of metabolic inhibitors(3) Acidity(4) Nature and concentrations of adsorbed metal cations(5) Species of moss

- Light significantly stimulated bisulfite loss from the external solution

- Differences in the degree of photoprotection between the two mosses might also explain their different abilities to detoxify bisulfite solutions

- DCMU inhibits photosynthetic electron transport and oxygen evolution. It caused a substantially reduced rate of bisulfite loss from the incubation solution, especially in R. triquetrus

- Experiment with DETC, an inhibitor of SOD, led to a very significant reduction in the rate of bisulfite loss from the incubation solution with both mosses

- Fe(III) catalysed extracellular oxidation of bisulfite as the pretreatment was accompanied by an increase in the sulfate concentration of the external solution.

- The reduced extracellular sulfate observed could indicate that Ca(II) enhances cellular uptake of bisulfite (indirect effect). - Ca(II) functions for stabilizing cell membranes or

embedded portein channels against loss of permeability control

- EDTA pretreatement did not cause lowered bisulfite disappearance

- Possibly, EDTA was relatively ineffective in chelating metals such as Fe(III) from the moss shoots.

- Alternatively, the EDTA may have disrupted normal membrane function so that bisulfite uptake or retention rates were modified

CONCLUSION

*Loss of bisulfite(1) External oxidation of bisulfite using metabolic (including photooxidative) energy

(2) ‘passive’ external oxidation of bisulfite catalysed by adsorbed Fe3+ ions

(3) cellular uptake and metabolic detoxification of bisulfite

AuthorsBhagawan Bharali

Jeffrey Bates

Department of Crop Physiology, Assam Agricultural University, Jorhat-785013, Assam, India and 2Division of Biology, Imperial College

London, Silwood Park Campus, Ascot, Berkshire SL5 7PY, UK