Adaptation Of Plasma Membrane H~+-ATPase And Apoplastic PH By Zinc Deficiency In Malus Hupehensis Rehd.Root

Zinc(Zn)is an essential micronutrient for all plants,participating in many physiological and biochemical processes.Zinc deficiency is common in apple production,showes obvious zinc-deficiency symptoms and affects yield and quality of apple fruits.The experiment was carried out with the seedlings of commonly used in production and the zinc-sensitive apple rootstock cultivar(Malus hupehensis Rehd.)under Zn deficiency stress with hydroponics.Not only the changing of the root morphology,roots activity and zinc concentration of Malus hupehensis Rehd.under Zn deficiency stress were investigated,but also the responses of plasma membrane H~+-ATPase hydrolytic activity,the kinetic characteristics of H~+-ATPase,PM H~+-ATPase enzyme concentration,PM H~+-pumpingactivity,net H~+ flux,ATP concentration,IAA concentration and apoplastic pH to Zn deficiency stress.We elucidated the relationship between apoplastic pH and PM H~+-ATPase in affecting M.hupehensis Rehd.root growth to adapt to Zn deficiency.The main results are as following:(1)The growth of M.hupehensis Rehd.was reduced under Zn deficiency.The M.hupehensis Rehd.seedlings were treated with Zn deficiency(0?mol·L-1)and Zn sufficient(4?mol·L-1).With the time of 4-week-old under Zn deficiency,the M.hupehensis Rehd.seedlings showed obvious zinc-deficiency symptoms under deficiency,for example the plant height were inhibited under zinc deficiency,the apex leaves were smaller and rosetted,shoot elongation was reduced,and seedlings grown had shorter roots than those grown Zn sufficient.The root morphology?roots activity and zinc concentration of Malus hupehensis Rehd.under Zn deficiency stress were investigated,the results indicated that the length,tip number,surface area and volume of roots of seedlings decreased under Zn deficiency conditions.It indicated that zinc deficiency stress inhibited the growth of roots.To the roots activity,the results showed that zinc deficiency stress decreased the roots activity,and then it decreased the zinc absorption by inhibited the growth of roots.(2)Zinc-deficiency affected plasma membrane H~+-ATPase activity in Malus hupehensis Rehd.root.Plasma membranes of M.hupehensis Rehd.roots were isolated using aqueous two-phase partitioning.The Purity of the seedling roots plasma membrane was above 85%.The activity of PM H~+-ATPase,hydrolytic activity of PM H~+-ATPase,proton pumping activity and maximum initial velocity Vmax were significantly lower under Zn deficiency than under conditions of Zn sufficient.The increase of ATPase Mechaelis constant Km indicated that Zn deficiency induced a decrease in the substrate affinity of PM H~+-ATPase.The immune-detection result showed that the enzyme concentration of plasma membrane H~+-ATPase activity was slightly lower under Zn deficiency.The result revealed that the lower hydrolytic activity of roots PM H~+-ATPase under Zn deficiency might be related to the decreased concentration of this enzyme under Zn deficiency.(3)The ATP and IAA concentration of M.hupehensis Rehd.was affected under Zn deficiency.The endogenous IAA contentration root and shoot apex were significant lower under Zn deficiency.The results indicated that zinc-deficiency stress decreased the IAA synthesis and inhibited IAA transportation from shoot apex to root.The ATP concentration under Zn deficiency stress was significant lower than Zn sufficient,it indicated that had a lower energy supply level in M.hupehensis Rehd.root under Zn deficiency,which affected the plasma membrane H~+-ATPase activity.(4)The net H~+ flux and apoplastic pH of M.hupehensis Rehd.root tips was affected under Zn deficiency.The net H~+ of M.hupehensis Rehd.root tips was efflux under Zn deficiency and Zn sufficient.The H~+ efflux in the Zn deficiency was significantly lower than that in the Zn sufficient.To the result of vanadate treat root tips,it indicated no obvious difference in net H~+ efflux between roots under Zn sufficient and Zn deficiency,and root pretreatment with vanadate eliminated any difference in H~+ extrusion.According to these findings,we suggest that PM H~+-ATPase might be involved in the regulation of H~+ flux under Zn deficiency.The apoplastic pH was significantly higher in the root tips of M.hupehensis Rehd.under Zn deficiency than under Zn sufficient.We conclude that Zn deficiency decreases plasma membrane H~+-ATPase activity,which most likely causes root cells to pump lower net H~+ into the apoplast,subsequently increased the apoplastic pH and reducing root development.