Functional Analysis Of Arbuscular Mycorrhizal Fungi In Apple Under Drought Stress

Apple?Malus×domestica?,because of its high economic and nutritional value,has become one of the most important cash crops,and has been widely planted in China and the world.The Loess Plateau is generally recognized as a eugenic region and a major producing area for apples.However,due to the low annual rainfall and extremely unbalanced distribution within the annual growth cycle,drought has become the most important factor that restrict the healthy and sustainable development of the apple industry in this region.Arbuscular mycorrhizal fungi?AMF?forms a symbiotic relationship with most terrestrial plants,which plays an important role in plant growth and development as well as adaptation to various stresses.It is discovered that,primary auxin-responsive genes Md IAA24,Md GH3-2 and Md GH3-12 were significantly induced by AMF.In this study,we used Malus hupehensis Rehd.seedlings inoculated with AMF and Md IAA24,Md GH3-2 and Md GH3-12transgenic apple plants to study the effect of different water supplies on seedlings,in terms of growth,mycorrhizal infection rate index,photosynthesis and chlorophyll fluorescence parameters,endogenous hormone content,activity of antioxidant enzymes and related genes expression.These results revealed the important role of AMF in response to drought stress in apple.The main results are as follows:1.AMF colonization could improve apple resistance against drought.Malus hupehensis Rehd.seedlings noculated with AMF were cultivated under well watered?WW?and drought stress?DS?conditions,respectively.Then the growth status was evaluated.The results showed that AMF and M.hupehensis formed a good symbiotic relationship,and the colonization rate was more than 60%.The plant height,root length,and dry weight between inoculated and non-inoculated plants exhibited significant differences.Under drought treatment,compared with the seedlings without AMF inoculation,AMF treatment could maintain higher photosynthetic parameters.Moreover,under drought stress conditions,the activities of peroxidase?POD?and superoxide dismutase?SOD?were significantly increased by AM inoculation,leading to relatively lower contents of O₂^-and H₂O₂.AM symbiosis alleviated the negative effects of drought on apple plants may by forming a better developed root system to absorb more water,by maintaining a better ability to balance the distribution of light energy in photochemical and non-photochemical processes,by activating the antioxidant enzymes to scavenge ROS and alleviating oxidative stress injury,by accumulating more soluble substances to improve osmotic regulation ability,or by up-regulating expression levels of stress-resistant genes.2.A total of 34 Aux/IAA genes were identified from the apple genome and 17 of them were cloned.A phylogenetic tree revealed that these proteins could be clustered into nine groups.There are four conservative domains among these Md IAA proteins by multiple sequences alignment.Many stress related cis-elements are present in most of the Md IAAs genes promoters.Expression analysis showed that these 17 Md IAAs also responded to drought,salt,low temperature and ABA treatments.Transgenic tobacco plants overexpressing?OE?Md IAA9 was subjected to osmotic stress treatment.Our results showed that the root lengths and fresh weights of the transgenic plants were significantly higher than those of the wild types under osmotic stress.The lower levels of REL and MDA and increased levels of proline and total chlorophyll in the transgenic lines indicated that Md IAA9 plays a significant role in enhancing resistance to osmotic stress.3.Md IAA24 expression is significantly induced by AMF inoculation.Overexpression of Md IAA24 improved apple drought resistance by positively regulating strigolactone biosynthesis and mycorrhization.Although symbiotic relationships were established both in the wild type?GL-3?and OE lines,the frequency of mycorrhiza in the root system of the two OE lines were significantly higher than that of GL-3.Gene expression analysis demonstrated that Md D27,Md CCD8a,Md CCD8b and Md MAXa in nonmycorrhized OE lines were significantly higher than those in nonmycorrhized GL-3plants.Moreover,the accumulation of SL in the two OE lines were higher than that of WT through the seed germination stimulation test.Furthermore,we observed that the OE lines were able to maintain a greater dry shoot and root weight under AM inoculation conditions.Under drought stress with the AM inoculation,the OE lines were less damaged,which was demonstrated by a higher relative water content?RWC?,a lower relative electrolytic leakage?REL?,a greater osmotic adjustment,a higher reactive oxygen species?ROS?scavenging ability,an improved gas exchange capacity and an increased Fv/Fm.Our findings demonstrate that the overexpression of Md IAA24 in apple positively regulates the synthesis of strigolactone and the formation of arbuscules as a drought stress coping mechanism.4.The presence of AMF significantly increased the expression level of Md GH3-2 and Md GH3-12.Knocking down the expression of Md GH3-2/12 reduced drought resistance by regulating strigolactone biosynthesis and mycorrhization.Although symbiotic relationships were established both in the GL-3 and RNAi lines,the frequency of mycorrhiza in the root system of RNAi lines were significantly lower than that of GL-3.Gene expression analysis demonstrated that Md D27,Md CCD7,Md CCD8a,Md CCD8b and Md MAXa in nonmycorrhized RNAi lines were significantly lower than those in nonmycorrhized GL-3plants.Moreover,the accumulation of SL in RNAi lines were lower than GL-3 through the seed germination stimulation test.Mycorrhizal transgenic plants showed increased sensitivity to drought stress compared with GL-3,as indicated by higher levels of REL and lower levels of RWC,osmotic adjustment,ROS scavenging ability,gas exchange capacity,Fv/Fm and abscisic acid.Taken together,these data demonstrate that Md GH3-2/12 plays an important role in AM symbiosis and drought stress tolerance regulation in apple.