Physiological, Biochemical And Molecular Changes Associated With Acquisition Of Desiccation Tolerance In Development Orthodox Seeds Of Jatropha Curcas

Dehydration is a natural terminal event for orthodox seeds of Jatropha curcuas resulted in dry seed with water content of 10-12% on fresh weight bases. Investigation of physiological, biochemical and molecular changes associated with natural dehydration process or after artificial dehydration of seeds at different developmental stages aimed to clarify the participation of two desiccation tolerance mechanisms (antioxitative mechanism and protein protective mechanisms) during seed development. The results concerning on hydrogen peroxide (H2O2) production and scavenging indicated to the decline in H2O2 production, consequently, lipid peroxidation product [as malondialdehyde (MDA)] during seed development. Activity analysis of main antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR), in endospermic and embryonic tissues showed a decrease in the total activity at the mature stage as compared with early stages. An exception was DHAR, which showed increased activity in mature seeds as compared to immature ones. Expression analysis of antioxidant enzymes genes at the RNA level during seed development confirmed the activity analysis pattern of studied enzymes and revealed that the decreasing in total SOD activity was related to the decrease in Cu/Zn-SOD gene expression, while the continuous activity of SOD during the maturation is related to an increase in Mn-SOD gene.Artificial dehydration resulted in increased SOD and DHAR activity, irrespective of the developmental stage. Analyzing the activity of antioxidant enzymes revealed poor participation of CAT and APX in H2O2 scavenging during the maturation of fresh seeds and in artificially dried developing seeds. No remarkable change occurred with regard to GR activities either during maturation or after artificial drying. Changes in SOD and GR isoenzyme patterns occurred during maturation-related drying. In contrast, artificial drying did not affect the isoenzyme pattern regardless of the developmental stage.The results concerning about the protein profile in Jatropha seeds showed an association between the acquisition of desiccation tolerance during seed development or after artificial dehydration and increasing in accumulated protein in both of total and heat shock proteins. Western blot analysis of dehydrins (DHNs) and small heat shock proteins (sHSP) demonstrate the association of two different DHNs and two different sHSPs with maturation and natural dehydration process. Quantitative analysis for the expression of two dehydrins genes (JcDHN-1 and JcDHN-2) and two sHSPs genes (JcHSP-1 and JcHSP-2) revealed a significant increase in expression of the examined genes during maturation and natural dehydration process. Also, a great increase in transcript amount of JCDHN-2 and JCHSP-2 was related to the drop in water content from 42% FW at mature seeds (90 days after flowering (DAF) to 10% FW at dry seed (100 DAF). Characterization of full length cDNA of expressed DHNs revealed to two types of DHNs. JcDHN-1 from type Y3SK2 with molecular weight 19.3 KDa composed of 175 amino acid residues and JcDHN-2 from type Y2SK2 with molecular weight 17.1 KDa composed of 156 amino acid residues. Characterization of full length cDNA of expressed sHSPs revealed that both proteins contain the alpha-crystallin domain (ACD) of alpha-crystallin-type small heat shock proteins. JcHSP-1 with molecular weight 24.43 KDa composed of 219 amino acid residues and JcHSP-2 with molecular weight 18 KDa composed of 157 amino acid residues.