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The atmosphere of the earth is divided into two layers on the basis of the presence of ozone, stratosphere and troposphere. The stratosphere is between 8 and 30 miles above the ground and we can not breathe any of its air. The ozone in this layer of air protects plants, animals, and us. Here it filters out photons with shorter wavelengths (less than 320 nm) of ultraviolet light (270 to 400 nm) from the Sun that would be harmful to most forms of life in large doses.This type of ozone is called "stratospheric" ozone.
Tropospheric ozone is the layer of air closest to the Earth's surface. The troposphere is the air from the ground to about 8 miles up into the atmosphere. We breathe air of this region. Ozone at the ground level is unstable. It reacts chemically with plants and the tissues of living creatures. It has the ability to irritate your lungs. Approximately half of tropospheric ozone originates from photochemical reactions involving nitrogen oxides, methane, carbon monoxide, and other substances. These gases are emitted through anthropogenic sources, mainly from combustion of fossil fuels and from some agricultural sources. The other half of the tropospheric ozone is produced from the downward movement of stratospheric ozone. The atmospheric lifetime of tropospheric ozone is about 22 days.
According to Intergovernmental Panel on Climate Change (IPCC) Report, Climate Change 2001, the concentration of carbon dioxide (CO2), methane (CH4) and nitrous oxide (N20) in the atmosphere has increased by 31%, 151% and 17% respectively since 1750. The total amount of ozone (O3) in the troposphere is estimate to have increased by 36% since 1750.
Troposphere ozone is considered as a pollutant by the World Health Organization and the United States Environmental Protection Agency (EPA). Ozone (O3) is regarded as one of the most damaging air pollutants to which plants are exposed (Thompson, 1992). Seinfel (1989) has measured concentrations as high as 400 to 500 ppb during period of severe pollution. In industrialized countries of the northern hemisphere, daily 8-h tropospheric O3 is estimated to have increased from approximately 10 nmol mol-1 prior to the industrial revolution to a current level of approximately 60 nmol mol-1 during summer months, and is predicted to increase 20% more by 2050 (IPCC, 2007). This is particularly relevant to agriculture because sensitive crops show a reduction in yield once the O3 exceeds 40 nmol mol-1 for extended periods (Heagle, 1989). Under favourable meteorological conditions, ozone may accumulate in the troposphere and reach a level that causes significant decrease in growth and yield of ozone-sensitive species in many parts of the world. The problem of phytotoxicity is well established in Europe (Jäger et al., 1992) and North America (Heck et al., 1988). Ozone a strong oxidant and it causes several types of symptoms including chlorosis and necrosis. It is almost impossible to tell whether foliar chlorosis or necrosis in the field is caused by ozone or normal senescence. Several additional symptom types are commonly associated with ozone exposure. These include flecks, stipples bronzing, and reddening. Ozone symptoms usually occur between the veins on the upper leaf surface of older and middle-aged leaves, but may also involve both leaf surfaces (bifacial) for some species. The type and severity of injury is dependent on several factors including duration and concentration of ozone exposure, weather conditions, and plant genetics. . Studies in open-top field chambers have repeatedly verified that flecking, stippling, bronzing and reddening on plant leaves are classical responses to ambient levels of ozone. Plants grown in chambers receiving air filtered with activated charcoal (CF) to reduce ozone concentrations do not develop symptoms that occur on plants grown in nonfiltered air (NF) at ambient ozone concentrations. Physiological processes such as carbon assimilation, translocation, nutrient acquisition are inhibited by ozone exposure that ultimately lead to suppressed plant growth and yield, but the question remains as to how this occurs. Heath and Taylor, 1997; Pell et al., 1997; Sandermann, 1998; Rao and Davis, 2001 have developed following hypotheses for these effects and the subsequent decline in plant productivity. (1) Membrane damage in leaf tissue results in ionic imbalances and other dysfunction. (2) Loss of photosynthetic capacity occurs due to lower levels of Rubisco activity and content. (3) A diminished ability to allocate carbohydrates to developing plant organs. (4) Production of signals leads to pathogen attack or wounding-type responses. (5) Accelerated senescence occurs. A number of recent studies utilizing molecular techniques, have significantly added to our understanding of how ozone affects plants, but many of the biochemical and molecular mechanisms involved in oxidant injury from ozone remain unclear.
We have studied the expression of caspase like gene (metacaspase) in maize plant treated by ozone. Caspase belongs to family C14 and clan CD, these proteases are intensively studied in animals because the regulate apoptic cell death. Their fame is also is a source of confusion because they hunt for caspase activities in plants resulted in the description of many "caspase like proteases" that are not probably related to caspases. Caspase like protease are defined as sharing sequence homology to animal caspases. Proteases that share sequence homology with animal casoase are absent in plant genome but plant have metacaspase. These caspase are unified in clan CD and use a catalytic Cys that is activated by catalytiv His for nucleophylic attack.Clan CD proteases are highly specific for cleavage after specific residues: Asp for animal caspase, Arg for metacaspase and Asn for vascular processing enzyme (VPEs). Caspase and metacaspase are usually cytoplasmic or nuclear. Metacaspase are produced with a linker protein that is proteolytically removed which results in the heterocoplex of a p20 and p10 chain. These chains have a relationship with animal caspase that have been suspected to be involved in PCD.
Metacaspases constitute a new family of caspase-related protein described by Uren et al. (2000). They have been identified in plants, fungi, and parasitic protozoa but are absent in mammals by bioinformatic analysis. Metacaspases are structurally related to caspases and show conservation of cysteine and histidine amino acid residues involved in the Cys-His catalytic dyad of the active domains of caspase. Caspases have been shown to play a central role in programmed cell death of mammalian cells, also called apoptosis. To date, no caspase gene has been identified in plants, yeasts, or protozoan parasites. However, since these organisms possess one or more metacaspases, it is conceivable that like caspases in mammalian cells, these caspase-related proteases could be involved in the PCD pathways in these organisms.
2 Materials and method
2.1: Ozone treatment and collecting of samples
Maize plants were grown in controlled condition. Ozone was diffused continuously at the rate of_____ .The maize plants were at distance of 2 and 3 metre away from ozone diffuser. Maize leaves were harvested at different stages and the samples were conserved at -80C°. We harvested older leave 10 and younger leave 12 respectively for our experiment because these leaves showed phenotypically ozone symptoms.
2.2: Designing of metacaspase primers
Physiological involvement of metacaspase have not studied so far in maize plant therefore
we designed metacaspase primers in order to study the expression of metacaspase in maize plants. Three types of metacaspase have been find in maize plants, metacaspase 83, 45 and B4. We designed primers for all these three metacaspase.
BT043382 --RKIALLVGINYPGTKAELKGCYNDVDRMRRCLVDRFGFDEADIRVLTDADRSAPQPTG 58
ACF83610 MGAKRAVLVGINYQGTKAELKGCHNDVARMRRCLVDRFGFDESGIRVLIDDG-SAPQPTG 59
ACG45179 MGQKRALLVGINYLGTDGELKGCLNDVARMRRCLVGRFGFDEADIRVLADADPSTPPPTG 60
BT043382 ANIRRALARLVGDARPGDFLFFHYSGHGTRLPAETGQHDDTGYDECIVPCDMNLITDQDF 118
ACF83610 ANIRRELARLVGDARPGDLLFFHYSGHGIRLPAETGKDDDTGYDECIVPCDMNLITDQDF 119
ACG45179 ANIRLELERLVAGARPGDALFFHYSGHGLQLPAETGEDDDTGYDECIVPCDLNLIKDQDF 120
BT043382 RELVQKVPEGCLFTIVSDSCHSGGLLDSAKEQIGNSTKQNKTQSREPDEPR----HSGSG 174
RAFIQ -----------------DSCHSGGLLDSAKEQIGNSTKQNKTQSREPDEPR----HSGSG 39
ACF83610 TELAQKVPSGCLFTIVSDSCHSGGLLDKTKEQIGHSTKQKQKQTQTQTQSRELEERSPSG 179
ACG45179 TDLVAKVPDGCRFTMVSDSCHSGGLIDKTKEQIGNSTKQNRTQQRREREMKP--PPPAPG 178
********:*.:*****:****::.* : : : . .*
BT043382 SGFRSFLKETVRDVFESEG-IHIPHSRR-------HGDD-DQDDGYAQPTGNGRTKNRSL 225
RAFIQ SGFRSFLKETVRDVFESEG-IHIPHSRR-------HGDD-DQDDGYAQPTGNGRTKNRSL 90
ACF83610 TSFREFLKGTVREAFESQG-IHLPHRSRSHQQGSGHGDDGDQESRYIN-TADAHVKNRSL 237
ACG45179 SALRVSLARIVRGVLESLGCIHCSRCR-------------VQQQGNSN---SSSISNRSL 222
:.:* * ** .:** * ** .: *:. : .. .****
BT043382 PLSTLIEMLKEQTGKDDIDVGSIRMTLFNIFGDDASPKIKKFMKVMLGKFHQGQSGEQGS 285
RAFIQ PLSTLIEMLKEQTGKDDIDVGSIRMTLFNIFGDDASPKIKKFMKVMLGKFHQGQSGEQGS 150
ACF83610 PLSTLIEMLKEKTGKDDIDVGSIRLTLFNLFKDDASPKIKKFMKVMLNKLQQGQHG---- 293
ACG45179 PLSTFIQMLRDKTGRHDVGVGSIRTTLFHHFGDEASPKVKRFMKAML------------- 269
****:*:**:::**:.*:.***** ***: * *:****:*:***.**
BT043382 GGGGVFGMVGALAQEFLKAKLEGKEEEAFKPALEQEVHSVDEVYAG-----SKAWAP-NN 339
RAFIQ GGGGVFGMVGALAQEFLKAKLEGKEEEAFKPALEQEVHSVDEVYAG-----SKAWAP-NN 204
ACF83610 ---GIVGFMGALAQEVLKAKLDGKEEEEFDPAMKQHVHSDQEVYAG-----TTARVP-SN 344
ACG45179 --LGKLRHDGKEAEEQSRVPREAEVEE----TLAQDAHSVEEVYAGPAAAAAAARVPPRN 323
* . * *:* :. :.: ** :: *..** :***** : * .* *
BT043382 GILISGCQTNQTSADATTPQGVSFGALSNAIQTILADKHG------KVTNKDLVMKAREL 393
RAFIQ GILISGCQTNQTSA---------------------------------------------- 218
ACF83610 GVLISGCQTDQTSADATTPKGVSYGALSNAIQAILAERG-------TVTNKELVLKARKM 397
ACG45179 GVLISGCQTDETSADATTADGMSYGALSNVIQTILAGDGKKRGVALAVTNRELVVRAREL 383
BT043382 LSKQGYTQQPGLYCSDEHVHVAFIC 418
ACF83610 LSKQGYTQQPGLYCSDENASAAFIC 422
ACG45179 LSRQGYTQQPGLYCSDEHATLPFIC 408
2.3: House keeping gene selection
The following genes Actin, S19 and Cyclophylin was studied at qPCR as a house keeping genes. Primers designed for each gene and their stability was analysed by geNorm.
2.4: RNA Extraction, reverse transcription, PCR and clonage
The extraction of total RNA (mRNA, tRNA and rRNA) from plant leaves was carried out by using the Rneasy Mini Kit (Qiagen) according to manufacture instruction. Complementary DNA (cDNA) were prepared by reverse transcription reaction by using omniscript kit (Qiagen).PCR at complementary cDNA was done by using specific primers for metacaspase and each gene of reference. Each PCR product was cloned in pGEM-T easy vector by thermal shock and cultured in E. coli bacteria for one night at 37C in the agitation of 150 rpm. Purification of overnight cultured bacteria was performed by using Miniprep kit (Promega).Purified plasmid DNA sent for sequencing to genoscreen laboratory in Lille (France).
2.5: Primers testing by qPCR
Melting curve analysis of designed primers of metacaspase and gene of reference was performed by qPCR. Melting curve analysis is used to identify the homogenity of DNA product. Even a single base change in heterogeneous DNA can change its melting temperature. qPCR was done by using loght cycler 2 Roche according to manufacturer instruction
3 Results and discussion
3.1: House keeping genes
Three housing keeping genes expression was studied by qPCR. All house keeping genes CP was converted into quantities. The quantity of each gene was put into geNorm software that tells us the stability of these genes.