Cassava postharvest physiological deterioration: a complex phenomenon involving calcium signaling, reactive oxygen species and proteome regulation

Abstract

The production of cassava (Manihot esculenta Crantz), the fifth most important starch crop worldwide after rice, wheat, maize and potato, is limited by many factors among which the short shelf life of its storage roots is a major constraint. This phenomenon known as postharvest physiological deterioration (PPD), is a syndrome attributed to a cascade of signalling events triggered by reactive oxygen species (ROS) at the wounded site of the tubers. The consequence is the rapid deterioration of cassava storage roots 24 – 48 hours after harvest, which renders the roots unpalatable and unmarketable. However, the molecular mechanisms and the interactions between different pathways involved in PPD in cassava are still poorly understood. This thesis focuses on investigating the interactions between Ca2+-calmodulin, ROS and proteome to fine-tune PPD process. An approach to delay PPD through the modulation of calmodulin gene expression and ROS activities using calcium and magnesium fertilizers was also assessed. Two cassava genotypes, South China 5 (SC5) susceptible and Qiong Zhong 1 (QZ1) tolerant to PPD were selected for this study. The experiment was performed at the Tropical Crops Genetic Resources Institute of Chinese Academy of Tropical Agricultural Science (CATAS) at Danzhou-Hainan (China). The activities of antioxidant enzymes of cassava storage roots during storage in PPD-susceptible (SC5) and PPD-tolerant (QZ1) genotypes were investigated at the time-points 0, 3, 6, 15 and 30 days after harvest (DAH). The analysis of their proteome changes using two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry (MALDI-TOF-MS/MS) was performed. In addition the effect of fertilizer containing calcium and magnesium fertilizers on antioxidant activities and PPD onset was tested. The results of the two-dimensional gel electrophoresis (2-DE) analysis demonstrated that there were 106 differentially expressed proteins (DEPs) in the two genotypes in the pairwise comparison of 3DAH/0DAH, 6DAH/0DAH, 15DAH/0DAH and 30DAH/0DAH from which 63 identified in QZ1 and 75 in SC5. The most DEPs were identified by MALDITOF-MS/MS as defense proteins (25 % and 23 %), carbohydrate and energy metabolismassociated proteins (19 % and 21 %), chaperones (14 % and 21 %) and antioxidant related proteins (13 % and 7 %) in QZ1 and SC5 respectively. PPD development also induced qualitative and quantitative changes in protein profiles, depending on the genotype. Western blot analysis of some expressed proteins confirmed the results of 2-DE. Antioxidant activities showed that cassava storage roots undergoing PPD are subjected to reactive oxygen species (ROS). High amounts of hydrogen peroxide (615.67 ± 3.41 μmol/g FW) in SC5 compared to QZ1 (252.38 ± 3.10 μmol/g FW) was observed at 6DAH corresponding to the date of the onset of PPD in SC5 confirming the implication of Hydrogen peroxide in PPD onset. Subsequently, the increased activities of ascorbate peroxidase, catalase (CAT), peroxidase and superoxide dismutase (SOD) were observed in PPD susceptible genotype SC5 at early stage of storage (3 to 6 DAH). The results suggest that SOD in combination with CAT could be the first line of defense against PPD to support PPD-tolerant cassava varieties. In addition, ethylene highly produced in SC5 (44.44 ± 1.80 ng/ml FW and 48.23 ± 1.37 ng/ml FW compared to 33.88 ± 0.42 ng/ml FW and 37.12 ± 0.32 ng/ml FW in QZ1 respectively at 3 and 6DAH) may act alone as an up-stream signal or simultaneously with CAT in response to the accumulation of H2O2 since a positive correlation was observed between these two components at a level of 5 %. The detection of calmodulin (CaM) at protein level coupled to the high relative expression level of its gene before the peak of hydrogen peroxide suggest it as a possible trigger of ROS during wound induced PPD in cassava storage roots (CSRs). The application of calcium and magnesium fertilizers delayed PPD onset for more than 10 days in the susceptible genotype (SC5); but magnesium fertilizer elevated PPD in the tolerant genotype by early onset at 6DAH in roots from plant treated with magnesium. In addition, Ca2+ fertilization led to a significant increase of H2O2 content (275 ± 0.6 μmol/g FW and 255.4 ± 0.01 μmol/g FW respectively in QZ1 and SC5 in roots from plant treated with calcium compared to the controls 245.8 ± 0.03 μmol/g FW and 263.6 ± 1.16 μmol/g FW for SC5 and QZ1 respetively. This increase of H2O2 was followed by an elevation of the activities of catalase and peroxidase in both genotypes, especially at harvest. Our findings suggest that soil fertilization with fertilizers containing calcium and magnesium may play a distinct role in PPD delay in cassava storage roots. These data further extend our knowledge about the chronology of the cascade of events occurring during PPD and the interaction between ROS production, calmodulin and proteome during PPD process. The results also provide new methodology to enhance PPD tolerance in cassava susceptible genotypes and reduce postharvest losses. Moreover, the results confirm that PPD is an active and complex process involving crosstalk between different pathways.