Determining Heat Tolerance Of Interspecific (Cucurbita Maxima X Cucurbita Moschata) Inbred Line Of Squash ’Maxchata’ And Its Parents

Plant confronts a multiple of biotic and abiotic stresses throughout its life. Heat stress is the most important yield limiting factor. Temperature is one of the main ecological variables that determine the natural geographical and seasonal distribution of plants. The elucidation of heat tolerance mechanisms is essential to combat the challenges of global warming. Cucurbits are consumed worldwide because of its high nutritional value and are included amongst the 10 leading vegetable crops. Over all, China ranks first among the cucurbits producing countries. Squash belongs to family Cucurbitaceae. Chinese squash (Cucurbita moschata) is best adapted to hot climate and is successfully cultivated in the tropical and subtropical regions, while the Indian squash (Cucurbita maxima) is widely grown in comparatively warm temperate areas of the world. C. maxima has very good consumer acceptability because of its excellent cooking quality and attractive brighter yellowish orange flesh color, but it is found to be heat and disease susceptible. As one of breeding strategy for squash improvement, an interspecific inbred line of C. maxima and C. moschata namely ’Maxchata’ was developed. So in this study,’Maxchata’and its parents were exploited in order to investigate the heat tolerance mechanisms in squashes.Photosynthesis is considered as the best diagnostic indicator of thermotolerance of plants because of its extreme sensitivity to high temperature. In this research work, first the extent of heat tolerance of newly developed interspecific inbred line of squash ’Maxchata’was determined through its photosynthetic attributes compared to its parents C. maxima and C. moschata. Plants of these three genotypes were subjected to three different temperatures i.e 30 C as control,37 C as moderate heat stress and 42 C as severe heat stress, for seven days. Results showed that various gas exchange attributes such as net photosynthetic rate (PN), stomatal conductance (gs), transpiration rate (E) as well as maximum photochemical efficiency of PSII (Fv/Fm) dropped significantly with increasing temperature, while intercellular CO2 concentration (Ci) increased showing the nonstomatal limitations. Further, chlorophyll pigments also degraded with heat shocks resulting in higher Chi a to b ratio and decreased chlorophyll to carotenoids ratio. However, these trends were more abrupt in C. maxima, chased by ’Maxchata’and then C. moschata. C. moschata had the best photosynthetic machinery to sustain the heat regimes, followed by "Maxchata’, while, C. maxima was the most susceptible.’Maxchata’with some degree of heat tolerance might have ability to cope with the climate change.Heat stress may disrupt the cellular homeostasis and cause severe impairments. Reactive oxygen species (ROS) have been found to play key role in the mechanisms responsible for these injuries. Plants have ROS scavenging and ROS avoidance mechanisms to maintain cellular homeostasis. Hence, cloning and characterization of some antioxidant enzymes and alternative oxidase genes were accomplished with the aim to broaden the genetic database of squashes and to examine the antioxidant and AOX genes responses of squashes to heat stress.Among the antioxidant genes CAT (CAT1, CAT2, and CAT3), SOD (Cu/ZnSOD, FeSOD, and MnSOD), and APX (APX1 and APX2) isozymes were cloned. The cloned members of AOX family of squashes included ’CmAOX2a,’CmAOX2b’,CmAOX2c’,CmAOX2d’and ’CmAOX2e’.Beside these genes, three other genes i.e. Actin, Chaperon 60(’CmCHN60’) and Cytochrome oxidase 2 (’CmCOX2’) were also cloned to support this study. The sequence analysis showed that these genes shared high sequence homology and conserved motifs with other orthologous genes.Next, the ultramorphological and some antioxidative changes induced in the leaves of ’Maxchata’and its parents upon exposure to moderate (37℃) and severe (42℃) heat stress were investigated to obtain insights into heat-tolerance indicators. The results showed that heat stress caused a relatively lower degree of membrane damage with lower malondialdehyde level and electrolyte leakage and higher proline contents in C. moschata and ’Maxchata’. The electron microscopy highlighted the maximum degradation of the leaf ultrastructure of C. maxima upon heat exposure. In contrast, C. moschata and ’Maxchata’exhibited lower degree of subcellular injury. The enzymatic activities of antioxidant enzymes i.e. superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (POD) were found to be the highest in C. moschata, moderate in ’Maxchata’, and lowest in C. maxima. In addition, the gene expressions of the CAT (CAT1, CAT2, and CAT3), SOD (Cu/ZnSOD, FeSOD, and MnSOD), and APX (APX1 and APX2) isozymes were unique in each of the studied squashes with increase in temperature indicating their differential contribution to heat tolerance. Although the selected genes were genetically similar in C. maxima and ’Maxchata’, most of the genes were more highly induced at elevated temperatures in ’Maxchata ’compared with C. maxima. Hence, the heat stress-induced gene expression in ’Maxchata’improved the efficiency of its antioxidant network and provided it some degree of thermotolerance.Further, the antioxidative alterations associated with heat tolerance in the stems and roots of the tested squashes were examined. C. moschata exhibited comparatively little oxidative damage, with the lowest hydrogen peroxide (H2O2), superoxide (O2-) and malondialdehyde (MDA) contents in the roots compared to stems, followed by "Maxchata". The enzyme activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD) were found to be increased with heat stress in tolerant genotypes. The significant inductions of FeSOD, MnSOD, APX2, CAT] and CAT3 isoforms in tolerant genotypes suggested their participation in heat tolerance. The differential isoform patterns of SOD, APX and CAT between stems and roots also indicated their tissue specificity.In the next part of the study, the role of alternative respiratory pathway was evaluated in the heat tolerance of squashes being the part of ROS avoidance mechanism. Plants of C. moschata, C. maxima and ’Maxchata’were sprayed with the inhibitor of the alternative respiratory pathway,2 mM salicylhydroxamicacid (SHAM) or water and were subjected to three different temperatures i.e control, moderate heat stress and severe heat stress, for seven days. Results indicated that the total, cytochrome and alternative respirations were higher in C. moschata and ’Maxchata’at both 37 C and 42 C, but decreased in C. maxima at 42 C. Moreover, ROS production was also enhanced upon heat exposure in SHAM treated squashes as compared to water treated ones. Furthermore, the transcript levels of APX1, APX2, CAT1, CAT2, CAT3, Cu/ZnSOD, FeSOD and MnSOD were determined in both the SHAM and water treated squashes that were exposed to heat stress. It was observed that CAT1 and FeSOD were downregulated in SHAM treated plants of C. moschata under heat stress conditions. While, the expression of CAT2 and MnSOD were lower in SHAM treated plants of both C. moschata and ’Maxchata. These results indicate some interrelationship of AOX and antioxidant systems.The genes expressions of the five genes/alleles of AOX2 were also analyzed in heat treated squash leaves to know about their heat-stress induced responses. Besides, their transcript levels were also determined in different parts of the young and mature squash plants grown at normal temperature to investigate the tissue-specific responses. ’CmAOX2a’and ’CmAOX2d’ were found to be highly induced in heat tolerant squashes to heat stress signals. These results suggest that alternative respiratory pathway may participate in increasing the thermotolerance of the squashes. Furthermore, the differential gene expressions of five CmAOX2 genes/alleles in different tissues and species suggest their tissue specificity and species specificity. Nevertheless, the higher expressions of most of these genes in flowers and fruit may confirm their specific role in these growth stages of plant development.These findings allow us to understand the role of some key members of antioxidant network and alternative respiratory pathway in squash heat tolerance and provide basic information to have deep insight into the complex mechanisms of heat tolerance in future that can be further exploited in squash breeding programs. Moreover, the cloning and expression analysis of AOX genes family in squashes can be a valuable addition for further research work in this area.