Biological Functions Of Aquaporins In Postharvest Citrus Fruit

Citrus is the most important fresh fruit in southern China.With the rapid development of citrus industry in recent years,postharvest production has become increasing important for realizing the annual supply of citrus and maintenance of fruit quality.As core elements of market competitiveness for citrus fruit,the appearance quality and internal quality are affected by various environmental factors during storage and transportation.Previous studies have revealed that postharvest water loss is the key abiotic factor causing quality deterioration and postharvest senescence in citrus fruit.Fruit water loss is not only closely related to environmental factors but also modulated by surface cuticle wax and water movement in fresh fruit.Water transport at the cellular level is regulated by aquaporins(AQPs),which are widely distributed in the inner membrane system(such as plasma membrane and tonoplast membrane)and participate in regulating water balance and stress response in plants.The differential expression patterns of AQPs during postharvest storage indicated that they affect the storage performance of citrus fruit,while the specific functions of citrus AQPs remain largely unknown.Here,a study of AQPs was performed by combining various molecular biological methods to systematically analyze the biological functions and related regulatory mechanisms of AQPs in response to postharvest water loss in citrus fruit(mainly ‘Newhall' navel orange fruit)during postharvest storage.The main results are as follows:1.Maintenance of high-humidity storage condition and treatment with the acidic solution and can alleviate postharvest water loss in citrus fruits.The relative humidity in the storage environment directly affects the postharvest water loss rate of citrus fruit.The low humidity storage condition accelerated the postharvest water loss and texture decline and increased the TSS content of fruits;High humidity storage condition can effectively delay water loss and maintain fruit quality.Besides,different acid-alkaline solution treatments affect the quality of citrus fruit during postharvest storage.Alkaline solution(pH = 9)accelerated the postharvest water loss and texture decline,while acidic solution(pH = 6)could effectively delay the postharvest water loss and maintain the fruit quality.These results indicated that maintain high humidity-storage conditions and treatment with the acid solution were effective strategies to alleviate water loss and improve the storage performance of citrus fruit.2.AQPs had spatiotemporal-and variety-specific expression patterns in citrus fruit during postharvest storage.A total of 39 AQPs were identified in the Citrus sinensis genome,which were divided into five subfamilies: plasma membrane intrinsic proteins(PIPs,12),tonoplast intrinsic proteins(TIPs,9),nodulin 26-like intrinsic proteins(NIPs,10),small basic intrinsic proteins(SIPs,4),and uncategorized X intrinsic proteins(XIPs,4).In different citrus varieties,the number and protein sequences of AQPs were conserved,while the expression patterns were differential.Most AQPs(including PIPs and TIPs)were down-regulated in the peel of tight-skin citrus(‘Newhall' navel orange and HB pomelo)and Satsuma mandarin fruit during postharvest storage,as well in the peel of Ponkan and Fairchild mandarin fruit.Notably,several members of NIPs were generally up-regulated in the peel of ‘Newhall' navel orange fruit under different storage conditions,and PIPs and TIPs were down-regulated.Besides,the expression pattern of AQPs was differential between the peel and pulp of citrus fruit during postharvest storage:most AQPs in the peel were significantly down-regulated at the initial stage of storage,while those in the pulp were down-regulated at the late stage of storage.These results indicate that AQPs have complex expression profiles upon water loss in citrus fruit,being time-specific in tight-skin varieties,tissue-specific between peel and pulp,and variety-specific between loose-skin and tight-skin varieties.3.CsPIPs were down-regulated by CsMYB96 to alleviate postharvest water loss in citrus fruit.CsPIP1;1 and CsPIP2;4 were two predominant CsPIPs highly expressed in the peel of citrus fruit,and their expression was continuously down-regulated during postharvest storage.Both CsPIP1;1 and CsPIP2;4 had water transport activity,and the water transport activity of CsPIP2;4 was much higher than that of CsPIP1;1.Besides,transient overexpression of CsPIP2;4 significantly increased the water loss rate of kumquat fruit.Notably,CsMYB96 specifically inhibited the expression of CsPIPs and activated that of wax-related genes(CERs and KCSs)to enhance the resistance of citrus fruit to water loss.These results indicate that the decrease in the expression of CsPIPs during postharvest storage is an important strategy to alleviate postharvest water loss in citrus fruit.Meanwhile,CsMYB96 was identified as a core regulator which simultaneously regulates CsPIPs and wax-related genes in response to water loss.4.CsNIP5;1 regulated the water balance of citrus by altering the membrane localization and transcription level of PIPs,and was regulated by Cs WRKY4 and Cs WRKY28: CsNIP5;1 was located on the plasma membrane in Xenopus oocytes and showed high water transport activity.In plant cells,CsNIP5;1 was located in the endoplasmic reticulum or donut-like structures.Furthermore,CsNIP5;1 altered the plasma membrane localization of PIPs through protein-protein interactions,leading to a decrease in cell water permeability coefficient.Besides,overexpression of CsNIP5;1 led to a significant reduction in the expression of multiple AQPs and promoted the accumulation of multiple osmotic adjustment substances(soluble sugars and proline),which enhanced the resistance to water loss.The transcription factors Cs WRKY4 and Cs WRKY28 were found to regulate CsNIP5;1,which was repressed by Cs WRKY28 at the initial stage of storage while activated by Cs WRKY4 at long-term storage.These results indicate that CsNIP5;1 enhances the defense to water loss by altering the subcellular localization and transcript level of CsPIPs;besides,CsNIP5;1 is regulated by multiple Cs WRKYs during postharvest storage.These results provide new insights into the process and regulatory mechanism of postharvest water loss in citrus fruit,as well as a theoretical basis for the maintenance of postharvest quality by genetic engineering and genetic breeding improvement.