Effects of Low Concentrations of Ozone (O3) on Metabolic and Physiological Attributes in Wheat (Triticum aestivum L.) Pants

Published by Oriental Scientific Publishing Company © 2018 This is an Open Access article licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-nc-sa/4.0/ ), which permits unrestricted Non Commercial use, distribution and reproduction in any medium, provided the original work is properly cited. Effects of Low Concentrations of Ozone (O3) on Metabolic and Physiological Attributes in Wheat (Triticum aestivum L.) Pants

Jeddah suffers from serious air pollution problems due to emissions from different sources such as industrial activities, oil refiners and desalination plants.There is a very poor legislation regarding emissions from old cars in streets as well from factories, which cause environmental hazards.Levels of heavy metals, gaseous air pollutants and particulate emissions far exceed internationally acceptable standards [1][2][3][4][5] .
Ozone concentrations in Jeddah were recorded to be between 40 -70 ppb6 [6][7][8] .These concentrations are high enough to affect many plant processes, such as photosynthesis, transpiration, nutrient uptake, senescence, changes in metabolic, biochemical and physiological processes, resulting in significant effects on crop growth and yield in Europe and the USA 9 .
Wheat is a nutritious and versatile crop, it has been known to be sensitive to O 3 Worldwide [9][10][11] .Chronic exposure to O 3 alters physiological processes leading to reduction in growth and yield of economic crops 1,8 .However, very little is known about its sensitivity in the Middle East.The information on impact of O 3 on plants in Saudi Arabia are extremely scanty and fragmented 7,8 , although high concentrations of O 3 were recorded 6 .
Amino acids are key elements in the balance between catabolism and biosynthesis of various compounds.Amino acids represent a source of energy, some amino acids are directly linked to antioxidant biosynthesis12 Wheat plant was chosen due to its economic importance, nutritional value and its compact size and rapid growth.
The aim of the present study was to clear our understanding related to molecular response of wheat (Triticum aestivum L) plants to chronic low levels of O 3.

Plant samples, growth conditions and Experimental design
Grains of wheat were washed with distilled water before soaking and were soaked in a continuously aerated distilled water in room temperature for 24 h in darkness to remove any pesticides.Then, 10-15 seeds were sown plastic pots (18 cm height x 16 cm diameter) filled with multipurpose compost at about 1cm from the surface.Each pot was watered with tap water until the first leaf expanded.Then plants were transferred to four closed fumigation chambers; 2 chambers were receiving 50 ppb O 3 for 8 h d -1 (9:00 am -5:00 pm) during Jan 2017 -March 2017 (Fig. 1).

Non-destructive harvest Visible injury symptoms
Foliar injury symptoms were assessed carefully, every week of each treatment by counting the number of injured leaves and estimating the percentage of each leaf's area showing injury (on a score of 0 'no injury' to 5 '100% injury') 1,3,8 .

Gas exchange measurements and chlorophyll content
Net CO 2 photosynthetic rate (A) and stomatal conductance (g s ) were measured using a portable LICOR (IRGA-LICOR-6400, Lincoln, NE, USA).Measurements were carried out the first true foliage leaf on weekly basis

Destructive Harvest Biomass and grain yield
As describe in our previous paper 3 , plants were selected from each treatment and divided into main organs every week throughout the entire course of the experiment for dry matter portioning.

Measurement of Photosynthetic Pigments
0.1g fresh weight of leaves was homogenized with pestle and mortar in in 10 ml acetone.The homogenate was centrifuge for 5 min at 9000 rpm in a cold room (5 0 C).The supernatant was used to determine the Photosynthetic pigments (chlorophyll a and chlorophyll b) using UVspectrophotometer (SHIMADZU 2500) 11 .Amino acids profile 0.1 mg sample was hydrolyzed with 6.0 M HCl at 110 0 C for 24 h, then samples were filtered and HCl was evaporated.Hydrolysates were dissolved in citrate-Na citrate buffer (0.1 M, pH 2.2) 13 .Levels of amino acids were measured by GC-MS (Shimadzu 2010, Japan).

Scanning Electron Microscopy
Leaf tissue was collected at the end of the experiment by taking leaf punches from the center half way between the leaf edge and midvein from the same leaves that were sampled for gas exchange.The leaf sections were fixed in 4% formaldehyde, 50mM PIPES pH 6.8 by vacuum infiltration for 20 min.The tissues were dehydrated and then critical point dried in liquid CO 2 .Surface images were taken on a JEOL 6060LV scanning electron microscope (JEOL USA, Inc., Peabody, MA, USA) 14 .

Data analysis
Data were log-transferred prior to analysis to ensure that they were normally distributed.One-way analysis of variance (ANOVA) test was applied to the log-transoformed data, differences between means were tested by Tuckey Test using SPSS statistical package, USA (Package 4, USA).

RESULTS AND DISCUSSION
Visible injury symptoms appeared in the form of chlorotic stippling on leaves.Ozone was found to increase number of injured leaves and degree of injury on leaves by 1.2-and 4.5-fold, respectively (Table 1).The effect of O 3 on net photosynthetic rates (A) started to appear on the third week of exposure (Fig. 2).
O 3 caused decreased in stomatal conductance (g s ) by 13.9, 12.1, 20.5, 22.7, 22.2 and 23.4% on the third, fourth, fifth, sixth, seventh and eighth week of exposure, respectively (Fig. 3).Chl b showed no significant response to or O 3 , while Chl a was decreased by 50% when compared to plants grown in filtered air (P Â0.01) (Fig. 4).
O 3 caused reductions in number of grains/ ear.Number of grains/ears, dry mass of grains by 18.6, 17,9% and 2-fold, respectively.A harvest index as indicated by 1000 grain weight, was decreased by 38.3% (Table 2).Moreover, O 3 caused decreases in protein content by 29.7% (Tab.2).
Proline was the only amino acid (AA) that showed increase in its level due to exposure to O 3 by 72.7%.On the other hand, O 3 caused decreases in levels of other amino acid analyzed in this study (table .3).The mean contents of phenylalanine (4.78%), isoleucine (3.53%), leucine (6.16%), valine (4.09%), lysine (2.80%) and threonine (2.89%) were found lower in plants fumigated with 50 ppb O3 when compared with those grown in FA (Tab.3).
The scanning electron micrographs demonstrate that leaf surfaces of intact leaves of wheat plants were greatly modified by exposure to 50 ppb O 3 .(Fig. 5).
In O 3 -treated plants, there was a considerable loss of turgor and collapse of epidermal cells adjacent to stomata.
The concentrations of O 3 used in the present study is similar to ambient O3 levels recorded in Jeddah 1 .This concentration is known to alter physiology of plants and reduce yield of economic crops worldwide.The extent of economic losses of O 3 to crops in this country has been poorly evaluated 3,9 .
The present investigation showed clearly that chronic exposure to low doses of O 3 (50 ppb 8hd -1 for 70 days) had negative effects on growth, biomass accumulation, and yield.Analyses of metabolic processes were performed in order to perceive O 3 toxicity at early stages.
The reductions in photosynthetic rates (A) are in agreement with our previous work 3 .Moreover, is in agreement with the general view reported in literature [14][15][16][17] .Stomatal conductance (g s ) was also reduced due to exposure to either ambient air or 50 ppb O 3 .The reduction in g s could lead to reduction in absorption of CO 2 and reduction in photosynthesis and finally in the growth and yield 3,18 .
The stomatal closure in wheat can be also attributed to increased concentrations of CO 2 in intercellular spaces (Ci) especially since the inhibition in photosynthetic rates was followed by inhibition in stomatal conductance.Therefore, the results of present study (supported with SEM micrographs) indicate that the changes in stomatal conductance in response to O 3 could be portioned into a direct effect of O 3 on the epidermal tissue and indirect effect via changes in photosynthetic process.
Growth and yield parameters and protein content showed inhibition in response to O 3 and this is in agreement with the results of other resercers [19][20][21] .The Changes in dry matter partitioning has an important implication under  The effect of O 3 on protein and amino acid contents is very complicated, and needs further investigations using more crops.However, O3 causes reductions in AA and protein which is reflected in poor growth and lower yield.

CONCLUSIONS
The present study indicated that O 3 stress caused a number of morphological and physiological changes in the wheat plants, including decreased shoot length, root fresh and dry weight and shoot fresh and dry weight and 1000-grain weight.
Exposure to chronic low doses of O 3 , negatively affected all the response variables studied, indicating, its detrimental effects on vegetative growth, biochemical activities and development.This study highlighted the gap of knowledge about the changes in assimilate partitioning.This warrants further investigation.

Fig. 5 .
Fig. 5. Scanning electron micrograph of leaf surface of wheat.Upper photo represent plants grown in FA, while the lower graph represent plants fumigated with O 3 (Magnification X 500)

Table 1 .
Effect of O 3 on foliar injury symptoms of Wheat leaves (Figures not followed by the same letter in each row are significantly different from each other at P ≤ 0.05, n=40).

Table 2 .
Effects of O 3 on yield parameters and protein content a Plants were harvested 70 d after exposure to O 3 (Legends as Table1).

Table 3 .
Response of amino acids profile of Wheat to O 3 Legends as Table1.