Studies On The Mechanism Of Tolerance Difference And Qtl Mapping In Response To Root-zone Hypoxia In Apple Rootstock Genotypes

Molecular oxygen (O2) is an absolute requirement for efficient production of ATP though oxidative phosphorylation in aerobic organisms. In eukaryotes, oxygen is the final electron acceptor in the mitochondrial electron transport chain. Higher plants are aerobic organisms requiring molecular oxygen to support respiration and various other life-sustaining oxidations. But they frequently experience a low oxygen (hypoxia) environment mainly due to soil waterlogging or complete flooding, soil compaction, or poor soil drainage. Hypoxia is considered as one of the major environmental stresses limiting plant growth and yield worldwide, especially in higher rainfall region areas. Apple (Malus domestica Borkh) is one of the most economically important fruits worldwide. But apple tree often encounters root-zone hypoxia stress in production, most notably due to transient flooding, soil and water mismanagement and use of heavy machine. Hypoxia stress in the root-zone is thought to be one of those important factors which restrict the development of apple. Therefore, more studies should be studied to explore the mechanism of damage and adaption in response to hypoxia stress in plant.Our previous study shown quite different tolerance hypoxia among Malus species was observed. M. sieversii is much sensitive to hypoxia but M. hupehensis was tolerant to hypoxia. However the differences in responses to root-zone hypoxia have not been well characterized between the two species. In this study, we comparably studied the root growth, photosynthesis, stomatal behavior, endogenous hormonal levels and mineral element of M. sieversii and M. hupehensis under normoxic and hypoxic conditions, and genetic analysis and QTL mapping of waterlogging tolerance was studied in an apple rootstock cross (Ottawa 3×Robusta 5)The main results were as follows.1. Root-zone hypoxia stress inhibited growth of seedlings of both Malus species, but with significant differences in intensity between genotypes originating from the two habitats. After 15 days'hypoxia treatment, the reduction in leaf number under hypoxia stress was more drastic in M. sieversii (21.0%) from semi-arid region, compared with M. hupehensis (8.1%) from humid region under hypoxia stress. The decrease in plant height and root length was also more in M. sieversii than M. hupehensis. There were significant differences in leaf number, plant height and root length between the controls and the treatments in M. sieversii. However, there were no significant differences for these measurements in M. hupehensis. At the final harvest, the shoot and root dry mass was reduced by 24.9% and 42.4% in M. sieversii and by 7.3% and 15.9% in M. hupehensis, respectively, as compared with the controls. In addition, there was significant decline in root/shoot ratio in both the Malus genotypes under hypoxia stress treatment as compared to control. However, M. hupehensis maintained higher root/shoot ratio (0.28) and M. sieversii suffered a sharp decline in root/shoot ratio (0.18). These differences in the growth characteristics may indicate different abilities to adapt to hypoxia between the two Malus species, which might be associated with their origins.2. Root-zone hypoxia stress inhibited root growth of seedlings of both Malus species, the total root length, root volume, the number of root tip, root surface area and root diameter of two Malus species under hypoxia stress was decreased, however, with significantly differences in intensity between two Malus species. After hypoxia treatment, the total root length of M. sieversii was reduced by 36.9%, while reduced by 18.6% in M. hupehensis, respectively compared with the controls, M. hupehensis showed the lower degree of growth inhibition. Different kinds of root length of M. sieversii were significantly reduced under hypoxia stress, except for 2.4².8 cm root. The root of 0.0⁰.4 cm in M. sieversii was reduced by 24.1% compared with the controls. The root of 0.4⁰.8 cm and 2.4².8 cm in M. hupehensis was significantly reduced compared with the controls, while the roots of 0.8¹.2 cm, 1.2¹.6 cm, 1.6².0 cm and 2.0².4 cm were increased, the roots of 0.0⁰.4 was decreased, but there was no significant differences between the stressed and control plants.3. Stomatal of two Malus species showed different changes in response to hypoxia stress. The stressed M. sieversii had rounder stomata and smaller stomatal slits than the control plants. In some cases, the stomatal slit of stressed new leaves appeared to be completely sealed. Moreover, stomatal density was increased in stressed new and mature leaves of M. sieversii compared with controls, stomatal density of new and mature leaves of M. sieversii were increased by 12.1% and 26.7%, respectively, as compared with the control. However, stomatal density was decreased between stressed and control M. hupehensis. In addition, little changes in stomatal length were found in the stressed and control plants.4. Pn decreased in response to hypoxia stress in both Malus species throughout the hypoxia period, but with significantly differences in intensity of two genotypes. The reduction in Pn was more drastic in M. sieversii (39.3%) than M. hupehensis (14.8%) under hypoxia stress, however, in M. hupehensis, a small decline in stomatal conductance was seen, this resulting in only a slight drop in photosynthesis. The reduction in Pn was accompanied with reductions in stomatal conductance in both Malus species, while the reduction in gs under hypoxia stress was more drastic in M. sieversii than M. hupehensis. At the same time, Light response curves for the two species were also established after 9 days of treatment. The reduction in Pn was accompanied with increase in light intensity in M. sieversii. However, Light response curves in stressed M. hupehensis were not significantly different from those of controls.5. Exposure to hypoxia altered the levels of endogenous hormones in leaves and roots in both Malus seedlings. Leaf and root abscisic acid contents increased in response to hypoxia stress in both genotypes despite different extents. Compared with M. hupenensis, M. sieversii was more responsive to hypoxia stress, resulting in larger increases in leaf and root ABA contents. The changes in leaf and root ABA contents correlating with the different tolerance of genotypes confirm the involvement of this hormone in plant responses to hypoxia stress. The immediate increase in leaf IAA content was observed in leaves of both Malus species in the first 3 days as compared to their controls, and then decreased to a lower level than that of the controls at 9 d. However, there were significant differences in IAA level between M. sieversii and M. hupehensis. Hypoxia stress treatment significantly increased leaf GAs content in both M. sieversii and M. hupehensis, but with differences in the pattern of accumulation between them. Increases in GAs content were observed in stressed M. sieversii and M. hupehensis at 12 and 6 days (26.9% and 32.3 increase, respectively) and levels in stressed plants remained very high until the end of the experimental period. In addition, zeatin riboside, dihydro-zeatin riboside, and isopentenyl adenine differ in their pattern of changes in both Malus seedlings under hypoxia stress. Based on variations in endogenous hormonal levels in both Malus species that are differing in their ability to tolerate hypoxia, we conclude that not a unique hormone but multiple hormones and their interplay are responsible for the hypoxia tolerance.6. Exposure to hypoxia stress altered the contents of 10 mineral elements (N,P,K,Ca,Mg,Na,Fe,Cu,Zn and Mn) in root, stem and leaf in both Malus seedlings, however, with significantly differences in intensity of different mineral elements and tissue between two Malus species. Root Mg,Na and P contents, leaf K and Zn contents and stem Cu, K, Mg and Na contents in M. sieversii increased under hypoxia stress, while root, stem and leaf Ca, Fe,Mn and N contents decreased. Root Na and Zn contents leaf Mn and Zn contents and stem Cu, Ca and P contents in M. hupehensis increased under hypoxia stress, while root, stem and leaf K,Fe,Mn and N contents decreased. These differences in the mineral elements may indicate different abilities to adapt to hypoxia between the two Malus species, which might be associated with their origins7. 194 F1 population derived from a cross between Ottawa 3 and Robusta 5 were used to s to identify quantitative trait loci (QTLs) controlling waterlogging tolerance of apple rootstock. Based on a linkage map of 306 SSR markers, a total of two QTLs were identified and mapped on two chromosomes by composite interval mapping (LOD≥3). One significant (LOD score of 4.2) QTL was identified and flanked by CHO3a09 and GD154₃ on chromosome 5, explaining about 21.4 of the phenotypic variation. The other QTL affecting waterlogging tolerance of apple rootstock was located on chromosome 13. The QTLs got in this study not only offered new information for understanding waterlogging tolerance of apple rootstock, but also provided a potential starting point for marker assisted selection of waterlogging-tolerance apple rootstock.