الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 197 |  |  |
| | | from | 197 | | |
Abstract
Barley is one of the most important cereal crops in the world and it ranks fourth among these crops after wheat, rice and maize. The benefits of barley are numerous, starting from being food for birds and animals and its role in industry up to its high nutritional value, such as containing betaglucans. Barley cultivated area is in increase worldwide. Egypt participated in this global production with approximately 131,890 tons, as reported by FAOSTAT (2015). Barley is capable of facing harsh stressful conditions, including hot, drought and salinity. The increasing of global temperatures considered as a stress factor for many cultivated crops. In this study, 23 days old seedlings of four Barley cultivars (G 129, G 135, G 134 and G 2000) grown at 18°C were exposed to heat stress at 35°C for 2, 4 and 8 hours compared with controls. In an attempt to understand the mechanisms of its resistance or adaptation to increasing thermal disturbances; some biochemical, physiological and genetical analysis were carried out by: 1- Physiological and biochemical examination of apparent responses to stress. 2- Monitoring the different responses of some heat stress related genes to heat treatments. 3- Examination of some enzymatic antioxidants activity under heat stress. 4- Computational analysis for HSP70, HSP90, HSP18, P5CS1, HSFA1 and HSFA2 protein structure. Physiological measurements, that estimated the rate of fat oxidation and infiltration rate showed the changes in MDA and Electrolyte leakage indicated that, the tow sensitive cultivars G129 and G135 showed the higher EC value comparing with the tolerant G134 and the moderate G2000. Complementary, the heat sensitive Cultivar G129 appeared the higher MDA value (means higher membrane damages), while the tolerant cultivar G134 show negative values of MDA. The tolerant cultivar G134 may have non-enzymatic antioxidant, which reduced the harm effect of oxidative effect of high temperature shock, while G134 was the more affected cultivar with the low temperature effect. All tested cultivars (Giza129, G135, G134 and G2000) showed slight decrease in chlorophyll A content for comparing with control. While, chlorophyll B was increased specially the cultivar G134 at HS for 4 hours. The content of Carotenoid decreased in the sensitive cultivar G129 in the first 4 hours of HS and then increased. Only the moderate cultivar G2000 appeared increase in carotenoid that may play a ROS scavenger as Non-enzymatic antioxidants. Total soluble protein banding pattern showed appearance of new bands after heat treatments in some cultivars, that indicates the expression of some genes under HS while absence in the other cultivars. For example, the sensitive cultivar G129 appeared a new protein band at 200 KDa in the treated seedlings compared with control. While the tolerant cultivar G134 showed increase for the total amount of soluble proteins with different banding pattern of the low molecular weight between 25 and 40 KDA. Analysis of proline content for the tested cultivars (G129, G135, G134 and G2000) showed increase values for all cultivars comparing with its controls. The tolerate cultivars G134 showed gradient increase in the proline content (in the line with level of P5CS1 gene expression). Even though the moderate cultivar G2000 shoed increase of proline content under HS treatment comparing with control, but it showed the lower proline content comparing with the other tested cultivars. Cultivar G2000 that appeared moderate tolerant to HS may has another components involved in tolerant mechanism, such as content of Carotenoids. The sensitive Cultivar G129 showed the maximum increase from the beginning of the HS treatment. The enzymatic antioxidant activity (SOD and CAT) which estimated using native PAGE showed that the response of tested barley cultivars to HS obtained only one type of SOD ( Cu/Zn- SOD) with two different Isozyme molecular weight at 22 to 24 KDa. Only the cultivar G-2000 appears second type of SOD (Mn-SOD) at molecular weight between 50 to 65 KDa. In spite of CAT activity, no cultivars appeared significant activity (60 KDa) even control or HS treatments. The expression differences of the selected genes related to heat stress tolerance (HSP70, HSP90, HSP18, HSFA1, HSFA2 and P5CS1) showed differential responses according the cultivar and heat treatment. Q RTPCR data shows different levels of gene expression of P5CS1gene for the HS treated Barley cultivars comparing with control seedlings. The tolerate cultivars G134 showed gradient increase in P5CS1 gene expression (in the line with the proline content). While the sensitive cultivar G-129 after 2h of heat shock did not appear increase in P5CS1 expression than its control, whereas its expression rose to more doubled after 4 hours of HS. Interestingly, the moderate tolerant cultivar G2000 appeared gradient increase in P5CS1 expression but lower than the rest of cultivars and this pattern was agree with its proline content. The levels of HSP70 gene expression showed no significant up regulation. Only the sensitive cultivar G135 treated 2h at 35o C, showed significant increase of expression rate about 15 fold comparing with the control, while the tolerant one G134 showed only 2 fold and 4 fold expression under 2 and 4 hours HS. Only the sensitive cultivar G129 appeared more than 70 fold of HSP90 expression after 2 hours of HS followed by the other sensitive cultivar G135 that appeared 20 fold after 4 hours of HS, while the tolerant and moderate ones did not show up regulation of HSP90. Quick and strong response from the sensitive cultivar G129 to HS whereas appeared up regulation of HSP18 gene (chaperon) with 170 fold expression. Comparing with the tolerant G134 that did not show up regulation for HSP18. This may indicate to the degree of harmful of heat shock on the sensitive cultivar. The tow studied HSFA1 and HSFA2 (transcription factors) showed significant expression with three fold only in the tolerant cultivar G134 under heat shock condition compared with control. The computational analysis using multiple databases and their tools is mentioned in the methods chapter. The nucleotide and protein sequences of Hordeum vulgare was compared with other plants i.e. Zea mays, Triticum aestivum, Oryza sativa and Arabidopsis thaliana. Some data analysis needed by addition some bacteria and fungus lines to show more details. After working on HSP70 ,HSP90,HSP18,HSFA1,HSFA2 and P5CS1 nucleotide and protein sequences, Hordeum vulgare appeared a far distance in the phylogeny relationships about the other studied plants. This result was clear after the data collection from ProtParam; which proved the differences in PH , Instability index, and amino acids ratios between barley and other plants. This result on the primary structure of proteins. As for the second protein structures , the MSA on JalView then using JPred appeared different in the number of alpha-helix and β- sheets, that with the increase of alpha helix it acquires more ability to stress tolerance. At the levels of Active Domain structure; the Prosite tool showed exchanged mutations among Barley and the other studied plants. The detected mutations did not affect the structure or function, whereas the secondary and 3D structures not changed. GC content for all studied genes in the selected comparative plants appeared higher GC ration in Barley, while plant grown in wide area with a lot of media changes needs that mutations for adaptation and natural selection. The previous findings point to the need of improvement of our local cultivars.