Alleviative Effects And Its Mechanism Of Exogenous Arbuscular Mycorrhizal Fungi(AMF) On Cucumber Seedlings Under Cold Stress

Low temperature can cause a range of injuries in plants and production of facility agriculture. Cucumber(Cucumis sativus L.)is one of the widely cultivated and thermophilic crops in the world. The researches of arbuscular mycorrhizal fungi(AMF)applying on horticulture have caused great attention in the world, they have played an important role in the functions on improving the absorption of nutrition, enhancing production and resisting abiotic stresses.In the present study, cucumber(Zhongnong No. 26)and AMF(Rhizophagus irregulari)are used as our plant genetics and inoculum. Cucumber seedlings with inoculation(AMF)and non-inoculation(NI)were subjected for two different temperature conditions: ambient temperature(25°C/15°C day/night) and cold stress(C)(15°C/10°C day/night). For exploring the physiological functions of AMF on improving cold-tolerance ability of cucumber, the testing indexes were including plant growth, net photosynthetic rate, photosynthetic fluorescence characteristics, antioxidant system, osmotic adjustment system, phytohormon content and main mineral elements content. Meanwhile, through applying transcriptome sequencing technology on inoculated cucumber roots, the results revealed the gene expression differences(DEGs) that induced by inoculation with AMF under cold stress; AMF molecular response mechanism in cucumber roots was also analyzed using transcriptome data, it was helpful for understanding the mitigative functions of AMF on cucumber in transcriptome level. From the RNA-seq analyzation results, we found the effects of AMF on DEGs belong to phosphate transport and abscisic acid(ABA)metabolism under cold stress; therefore we investigated the influence of inoculation on plant phosphorus uptake and transport as well as abscisic acid metabolism under cold stress. The main research results were as follows:1. The growth of seedlings and infections of arbuscular, vesicular and hypha were inhibited by cold stress. However, symbiosis with AMF significantly improved the plant growth on roots part, and the content of chlorophyll, net photosynthetic rate, and PSII photochemical activities were increased in the cucumber seedlings leaves. Indicated that AMF had helped plants offset the inhibition on photosynthetic electron transportation, the stomatal and non-stomatal limitation caused by cold stress. Under cold stress, the H2O2 content was increased in seedlings leaves, the imbalance of reactive oxygen species(ROS)could lead to aggravate the membrane lipid peroxidation, but inoculated by AMF helped to alleviate the membrane lipid peroxidative injury by enhancing the activity of SOD and CAT, in order to maintain the balance of ROS level. Content of phytohormone was significantly influenced by cold stress, however, this symbiosis relationship would effectively helped to regulate the balance of phytohormone, enhanced the content of GA3, ZR and BRs on plant leaves and decreased the IAA levels. They also helped the absorption of mineral nutrition from soil and improved the growth of plants, like N, K, Zn, Fe, and Mg. In conclusions, inoculation with AMF had positive influence on plant physiological function, finally relieved the damage cold-induced injury on cucumber seedlings.2. To determine the molecular mechanism of AMF in cold resistance, the present study performed transcriptome sequencing of cucumber roots. First, for the mRNA-Seq data, nearly 67 million(87.5%) reads were mapped to the cucumber genome, whereas only 758,106(0.91%) and 281,324(0.34%) reads were mapped to the AMF genome for treatments AMF and AMF+C, respectively. The findings indicated a low proportion of AMF mRNA in the roots. Second, compared to the NI+C treatment, AMF+C had 2,173 differentially expressed genes(DEGs) of cucumber genes, of which 1,128 were up-regulated and 1,045 were down-regulated. Interestingly, we detected 180 cucumber DEGs, which were significantly down-regulated by cold stress, but significantly up-regulated by AMF inoculation under cold stress. We inferred these 180 cucumber genes as key cold-resistant genes that were induced by AMF under cold stress. Third, functional enrichment analysis of cucumber DEGs between NI+C versus AMF+C showed that these mainly participate into the biological processes of oxidative metabolism and ion(nitrate and iron) uptake/transport. Together, our findings suggest that AMF-induced key genes respond to cold stress and provide clues for further functional analysis, which may improve our understanding of the molecular mechanism underlying the role of AMF in strengthening cold resistance in plants. Most of difference expression genes(DEGs)of AMF in cucumber roots were down-regulated, and there was significantly difference in the expression of the genes of glycerol-3-phosphate catabolic process and NADH oxidation in biological process, thereby, these genes might influence the symbiosis relationship with plant and function of AMF under cold stress. Besides, the expression of predicted AMF specificity cucumber phosphate transporter(predicted CsPT1-11) was also highly enhanced by inoculated with AMF, which demonstrated that phosphate uptake and transportation were enhanced by inoculation in cucumber roots maybe by inducing the expression of predicted CsPT1-11 gene. In addition, through GO annotation and KEGG passway enrichment analysis, abscisic acid metabolism genes were significantly changed in inoculated treatments under cold stress.3. Symbiosis with arbuscular mycorrhizal fungi(AMF)could improve plant phosphorus(P)uptake and alleviate environmental stresses. Along with transcriptome results, it could be also an effective mean to promote plant performance under low temperature. The combined effects of arbuscular mycorrhiza and low temperature(15°C/10°C day/night)on cucumber seedlings were investigated. Succinate dehydrogenase and alkaline phosphatase activity in the intraradical fungal structures and expression profiles of four cucumber phosphate(Pi)transporters, the fungal Pi transporter GintPT and alkaline phosphatase GintALP were determined. Cold stress reduced plant growth and mycorrhizal colonization. Inoculation improved cucumber growth under ambient temperature, while under cold stress only root biomass was significantly increased by inoculation. AMF supplied P to the host plant under ambient temperature and cold stress, as evident in the higher P content of mycorrhizal plants as compared to non-mycorrhizal plants. Thus, the cold-stressed cucumber seedlings still benefited from mycorrhiza, though the benefit was less than under ambient temperature. In accordance with this, a cucumber Pi transporter gene belonging to the Pht1 gene family was strongly induced by mycorrhiza at ambient temperature and less under cold stress. The other three tested Pi transporters of different families were most highly expressed in cold-stressed mycorrhizal plants, suggesting a complex interactive effect of mycorrhiza and cold stress on internal P cycling in cucumber plants.4. Under cold stress, non-inoculated plants had higher ABA content, but it was lower in inoculated plants, and had no significantly influence on ABA content on cucumber roots. The shoots and roots growth were significantly increased by exogenous ABA in inoculated plants under cold stress, but with the treatment of ABA inhibition—NDGA exogenous treatment, the positive role of inoculation with AMF on plant root growth was inhibited, which indicated that, accompany with AMF, ABA might also play positive role in helping cucumber to relieve growth slowly problem causing by cold stress. The expression of ABA induced genes MYB and MYC on inoculated plants, and they were enhanced slowly after spraying with ABA under cold stress, ABA-induced genes were activated by NDGA, which might be because exogenous ABA weakened the response of cold stress on inoculated plants. However, the ABA response of non-inoculated plans seemed more sensitive to cold stress, and their expression was changed with the inoculation by AMF under cold stress; at the same time, exogenous ABA significantly induced the expression of ABA important synthetic gene CsNCED2 and catabolite gene CsCYP707A1, and the expressions of ABA dissociation genes CsBG1 and CsBG2 were highly enhanced on inoculated plants by exogenous NDGA, which indicated that, under cold stress, genes expressions of ABA metabolism was depended on type of gene and treatment with inoculation.