While numerous studies explored the response of walnut plants to drought stress (DS), there remains a significant gap in the knowledge regarding the impact of heat stress (HS) and the combined effects of DS and HS on the recovery capacity of walnut trees. This study aimed to investigate the mechanism of Persian walnut (cv. Chandler) response to the combined DS and HS, focusing on various aspects including photosynthesis, water relations, and osmotic regulation. The treatments involved subjecting plants to DS (through a withholding method for 24 d), HS (gradually up to 40 °C for 8 d), and a combined DS and HS, which were compared to a control group (no stress) during the stress and recovery phases. The results showed that DS had significantly more negative effects on chlorophyll content, relative water content (RWC), leaf water potential (WP), osmotic potential (OP) compared to HS. Involvement of osmoregulation mechanism was detected more in DS and HS plants through the accumulation of proline, glycine Betaine and total soluble carbohydrates. The functionality of photosynthesis was significantly impacted by both HS and DS, respectively. While the HS accelerated the change of the abovementioned physiological processes in drought-stressed seedlings. Consistently, more pronounced damage was found in leaves under the combined stress, alongside the decrease RWC, chlorophyll content and fluorescence ratios. Based on the analysis of the linear mixed-effect model, the effects of combined stress and HS on photosynthesis parameters were detected in the early stages of stress compared to DS. Within a range of stresses, the abovementioned physiological processes of individual and combined-stressed plants recovered to levels comparable to those of the control. Our results also showed a substantial reduction in the expression of the photosynthetic genes (Fd, Cyt b6f, and PsbB) in Persian walnut saplings under abiotic stress conditions indicating significant damage to their photosynthetic apparatus. This study highlights that, under scenarios of aggravating drought occurring with heat, walnut seedlings could face a high risk of damage to physiological structures in relation to the synergistically increased hydraulic and thermal impairments.
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