Characteristics of the response of Actinidia polygama to long-term waterlogging stress

Abstract: 【Objective】Kiwifruit is a worldwide fruit with delicious taste and rich nutrient substances.However, kiwifruit tree is sensitive to waterlogging, which severely limits kiwifruit production in southern China. Developing effective strategy to cope with waterlogging stress is urgently needed for development of kiwifruit industry. Grafted kiwifruit seedlings are widely used in the production of kiwifruit and waterlogging tolerant rootstock is a key strategy to deal with the stress.‘Zhemizhen 1'is an excellent strain of Actinidia polygama (Sieb. et Zucc) Maxim., which has a flourishing root system and a high stress tolerance. In the present study, artificial waterlogging was used to test its effects on physiological traits and stress related genes expression in order to address the mechanisms of the response of kiwifruit to long-term waterlogging stress.【Methods】In vitro plantlets of‘Zhemizhen 1'were used in this study. After expanding propagation and rooting culture, plantlets were transferred to medium with normal water and fertilizer management. Plants with eight leaves and uniform size were chosen for waterlogging treatment, in which water was irrigated and maintained 3 cm above the soil surface. The first sampling was carried out prior to the treatment. The second sampling was taken three weeks later, and then samples were collected every ten days, with a total of five sampling times (0 d, 22 d, 32 d, 42 d and 52 d) . Chlorophyll content was measured by SPAD-502. Data related to plant morphological traits including shoot weight, root weight, root length, root number, shoot height, root activity, aerial root, stomatal density, and stomatal length and width were collected. The activities of key enzymes e.g. SOD and POD were analyzed and the contents of H2 O2 and MDA were determined in each sample. RNA was extracted from fresh kiwifruit leaves and cDNA was synthesized by reverse transcription immediately.Based on previous reports, some stress signal genes (AcCIPK9, AcCIPK13, AcERF4 and AcERF5) and stress related functional genes (AcADH, AcHB1, AcPRT6, AcPDC1, AcSAD, AcGAD, AcHSP17.5 and AcLBD) were screened from the kiwifruit genome. Quantitative real time PCR was carried out to determine the expression levels of these genes in‘Zhemizhen 1'under waterlogging stress. Variance analysis of related data was carried out by SPSS 17.0 software.【Results】During the long-term waterlogging treatment, the chlorophyll content of‘Zhemizhen 1'did not change so much at the beginning (22 d to42 d) , but significantly dropped after 52 days. In the early treatment stage, the root/shoot ratio decreased significantly, and reached a lowest value at day 32. Thereafter, the ratio increased remarkably. The formation of aerial roots was observed before the second sampling time. Later on, the number and the activity of aerial roots increased quickly, which contributed to the high tolerance of‘Zhemizhen 1'to waterlogging stress. When waterlogging happened, some of the stomata closed. By 22 days from the treatment, the stomatal density had increased significantly. However, after day 22, there was no more increase in stomatal density, which is another aspect of the high adaptability to waterlogging stress. The higher contents of H2 O2 and MDA indicated that‘Zhemizhen 1'had been damaged by waterlogging.Their decrease at day 52 might be related to the relatively high activities of SOD and POD. On other hand, the expression levels of stress related genes changed in‘Zhemizhen 1'under waterlogging condition. The genes of AcCIPK9, AcERF4 and AcERF5 responded slowly in the early stage. They all reached the highest level at 42 d. AcCIPK13 was also repressed in the early stage and increased later.AcSAD, AcGAD, AcHSP17.5 and AcLBD were highly induced by waterlogging stress, indicating that their products might protect the plant. AcADH, AcHB1, AcPDC1 and AcPRT6 are members in the hypoxia response network. AcADH and AcPDC1 showed similar expression pattern, maintained low on22 d and 32 d, and then significantly enhanced later on. AcPRT6 was noted as a negative regulator of the other hypoxia genes and its expression was high on 22 d but low in the rest of time.【Conclusion】During waterlogging treatment, ‘Zhemizhen 1'produced aerial roots quickly with increased high root activity. Meanwhile, adjustments of root/shoot ratio and the stomatal distribution help kiwifruit to survive stress condition. With the high activities of antioxidant enzymes and enrichment of stress tolerant proteins during waterlogging stress, ‘Zhemizhen 1'showed a strong waterlogging tolerance. Therefore, ‘Zhemizhen 1'can be used as a waterlogging tolerant rootstock.