| Alkali stress is one of the main stresses that affect the agriculture development,leading to a serious reduction of yield and quality of crops.The soil pH value in apple eugenics area--loess plateau production area in China is mostly higher than 8.0,which is severely affected by soil alkalization.In previous study,Fupingqiuzi(Malus prunifolia Brokh.)was identified as an alkali-tolerance rootstock resource.To our knowledge,root is the first organ to sense the pH changes of soil environment,and proton pump(plasma membrane H~+-ATPase)is able to respond to a variety of abiotic stresses with multiple physiological functions.Therefore,the study on the response mechanism of proton pump in roots of M.prunifolia under alkali stress to the explore of the reasons for its strong alkali tolerance will provide the theoretical reference and technical basis for the study of alkali tolerance and further mining alkaline-tolerant rootstock resources of apple.In this study,the method of tissue culture and hydroponic culture were used.We used the hydroponic culture seedlings of M.prunifolia with the same genetic background to measure the changes of leaf photosynthetic characteristics,root electrical conductivity,dry and fresh weight,root activity,proton secretion,and proton flow rate of M.prunifolia seedlings under alkali stress.Bioinformatics analysis of plasma membrane H~+-ATPase gene family of Malus domestica was also performed.The relative expression of different plasma membrane H~+-ATPase family genes under alkali stress at different time was quantitatively analyzed by qRT-PCR method.So our study investigated the mechanism of alkali tolerance of M.prunifolia from plant physiology level to molecular biology level.Studies showed that:1.After 7 days’alkali stress treatment,the upper young leaves of M.prunifolia seedlings began to appear yellowing symptoms and the color of the lower leaves was normal,while in the control group the upper young leaves were normal green.On the 13th day of alkali stress treatment,the chlorophyll content decreased about 30%,Pn and Gs of mature leaves in the treatment group showed remarkable reduction compared with the control group;and the dry/fresh weight(biomass accumulation)of aboveground plants in the treatment group was lower than those in the control group,but did not reach a significant level;the electrical conductivity of roots in the treatment group showed a significant upward trend;the root activity decreased by 16%and the fresh weight decreased significantly,but the dry weight didn’t show the significant variation compared with the control.2.Within three days alkali stress treatment,the flow rate of H~+in different areas of the root system of M.prunifolia seedlings was negative,and the influx rate of H~+was greater than that of secretion,and after 3 days of treatment,the H~+flow rate of root tip,meristem zone(200μm),elongation zone(400μm)and root hair zone were positive,indicating that H~+secretion was larger than that of influx flow at this time in order to maintain the stability of pH value in the environment.The H~+flow rate reached the maximum on the 7th day of treatment,and tended to be stable efflux.After 13 days alkali stress treatment,the proton secretion experiment showed that the color of the culture medium around the root system became yellow,indicating the proton secretion;and there were no significant changes in the color around the root system of the alkali treatment group after adding proton pump inhibitor sodium vanadate;yet there were almost no change in the color of the culture medium around the roots of the control group before and after adding sodium vanadate.It is suggested that under the alkali stress the proton pump in the root system of M.prunifolia alleviate the damage of alkali through promoting H~+secretion.3.The results of screening,comparison and analysis of the proton pump gene sequences of on GDR database demonstrated that the plasma membrane H~+-ATPase family contained 13 genes,distributed on 10 chromosomes,and the structures were relatively conservative.Subcellular localization was predicted on the plasma membrane,and there were similar trans-membrane structures and the same trans-membrane times.The results of qRT-PCR analysis on various tissues and organs of M.prunifolia seedlings showed that 13proton pump genes were specifically expressed in stems,buds,young leaves and mature leaves of M.prunifolia,and the expression of MdAHA11 was higher in roots.The expression of MdAHA10 was higher in stem,MdAHA5 was higher in stem and leaf,the expression levels of MdAHA1,MdAHA2,MdAHA3,MdAHA7 and MdAHA12 were very low in all tissues,and it’s nearly undetectable;compared with the control group,the expression levels of MdAHA4,MdAHA5,MdAHA6,MdAHA8,MdAHA9,MdAHA10,MdAHA11 and MdAHA13 were all within the range of 1.5 folds under alkali stress. |
