Protein Preliminary And Photosynthetic Physiology Of Exogenous Brassinolide To Improve Hypoxic Tolerance In Cucumber Seedlings

Plants frequently experience limited oxygen availability, mainly due to mismanagement in production practice of soilless culture and soil flooding. The growth of plant was inhibited under hypoxic stress. Cucumber (Cucumis sativus L.) is one of the important vegetables in world, and major protected cultivation vegetable crops in China. Cucumber roots are weak and senilism, so they require higher rhizosphere oxygen cucumber is highly sensitive to hypoxia. Brassinolide (BR) has been recognized as a new kind of phytohormones and played an essential role in plant development and stress resistance. Previous researches suggested that BRs could regulate respiration, carbon metabolism and antioxidant system activity of cucumber seedlings under hypoxic stress. However, reports about the effects of brassinolide on photosynthesis and protein in cucumber seedling under hypoxic stress were limited.Cucumber(Cucumis sativus L.)’Zhongnong6’ was applied in our study, and exogenous24-epibrassinolide (EBR) was added in nutrient solution. The influence of EBR on protein expression, enzyme activities and photosynthesis in cucumber under hypoxic condition was studied, and the physiological mechanisms of cucumber responds for hypoxia were investigated. The results are as follows:1. Effects of exogenous brassinolide on the growth and soluble protein expression in cucumber seedlings under hypoxic stressHypoxic stress significantly decreased the cucumber growth and soluble protein content in roots, but increased in leaves. EBR could alleviate the growth inhibition in cucumber seedling which induced by hypoxic stress with increasing longer roots, dry weight and root tips. Meanwhile, exogenous EBR inhibited the changes of growth and soluble protein content under hypoxic stress. Hypoxic stress induced a new protein (relative molecular weight63.96×103) in cucumber roots and increased the abundant of eleven proteins at least. However, Exogenous EBR could significantly decrease the expression of these proteins and markedly affected nine protein bands in leaves. Therefore, it was possible that these proteins have relations with exogenous EBR alleviating the effect of hypoxic stress in cucumber seedlings. Thus, we considered exogenous EBR could alleviat the inhibition of hypoxic stress on cucumber seedlings by changing the soluble protein content and expression.2. Effects of exogenous brassinolide on protein expression of respiratory metabolism and antioxidant system in cucumber seedling roots under hypoxic stressPyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH) were induced by hypoxic stress. After application of EBR for three days, the expression of PDC and ADH isozymes increased by18.8%and28.8%compared to the hypoxic treatment. However, the abundance of PDC, ADH and LDH isozymes decresed by19.5%,25.6%,53.4%and26.4%,26.0%,28.4%on the6th and9th day, respectively. Hypoxia enhanced the expression of succinate dehydrogenase (SDH), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), malic enzyme (ME) isozymes, and after application of EBR at6th and9th, IDH and MDH isozymes were significantly increased but SDH isozymes was decreased. UGPase、ALD were induced by hypoxic stress, the abundance of UGPase and ALD incresed by20.50%,30.80%and47.81%,56.17%on the3th and6th day, respectively. Compared to the hypoxic treatment, the abundance of ALD at3th and UGPase at6th after application of EBR was significantly increased by11.20%and22.15%, respectively. Compared to control, the SOD, POD and CAT isozymes of cucumber seedlings under hypoxic stress treatment were decreased at3th,6th and9th days. However, BR increased the expression of SOD isozymes by5.22%and10.25%on3th day and6th day compared to hypoxic treatment and POD isozymes were also increased13.81%and9.46%, respectively. But CAT isozymes were not changed significantly.The effects of EBRs on the gene expression of respiration and antioxidant in cucumber seedlings under short-term hypoxic stress suggested that the gene expression of ldh, pdc, idh, sdh, mdh, ald, ugpase, pfk, hk, pod increased and cat, sod and fk decreased at3th day of treatment. After application the EBR, the expression of ldh、idh and pfk were further increased but the pdc, sdh, mdh, cat, sod and ugpase had a slight decreased. Our results suggested that hypoxic stress inhibited the growth of cucumber seedling roots and EBR might regulate the isozyme expressions of anaerobic respiration, aerobic respiration and antioxidant enzymes of cucumber roots thereby alleviated the suppression effects of hypoxic stress.3. Effects of exogenous brassinolide on the mitochondria antioxidant system, ATPase activity and cellular ultrastructure in cucumber seedling roots under hypoxic stressUnder hypoxic stress, the superoxide anion (O2-) production rate, hydrogen peroxide (H2O2) content and malondialdehyde (MDA) contents were significantly enhanced, and root and ATPase activity were decreased resulting in cell damage. Compared with the control, activities of SOD, POD and CAT were induced and were higher at3d, and significantly lowered at6d by hypoxic stress. However, the treatment of EBR could keep higher activities of SOD, POD and CAT for a longer time, reduce the generation of O2, H2O2and MDA contents and significantly increased the root and ATPase activity, injured of root tip cell was released. The results indicated that exogenous EBR raised the activities of mitochondria antioxidant enzymes, decreased the O2-production rate, H2O2and MDA contents. Therefore, exogenous EBR might improve the mitochondria antioxidant system, ATPase activity and cellular ultrstructure of cucumber seedlings under hypoxic stress thereby alleviating the adverse effects of hypoxic stress.4. Effects of exogenous brassinolide on photosynthesis, poly amines and inoroganic ions content in cucumber seedlings under hypoxic stressWhen exposed to hypoxic stress, net photosynthetic rate (Pn), apparent quanta yield (AQY) and carboxylation efficiency (CE) were decreased significantly, and quantum efficency of open PSⅡ reaction center (Fv’/Fm’), photochemical quenching (qP), PSⅡ photochemisty rate (ΦPSⅡ), however the raletive electron transport rate(rETR)in leaves of cucumber seedlings were significantly decreased, but the allocation of antenna heat dissipation energy (Drate), and non-photochemical quenching(qN) were significantly increased, and the cellular ultrastructure was abnormal, which indicated that hypoxia caused photo-inhibition to cucumber seedlings. However, EBR could alleviate or release the damages in chloroplast and mitochondria under hypoxic stress, and enhanced Pn, Fv’/Fm’, qP, rETR, PSⅡ and Prate. The results suggested that EBR could play a positive role in alleviating damage in mitochondria and chloroplast ultrastructure and photosynthetic characteristics, and make the leaf keep a higher photosynthetic performance, and alleviate the damage of hypoxic stress.Hypoxic stress significantly increased the contents of polyamines including Put, Spd, Spm, total PAs and Put/PAs ratio in leaves and root, and decreased (Spd+Spm)/Put ratio. The change of root was greater than that of leaf. K+, Ca2+, Mg2+, Fe3+, Zn2+and Mn2+content in leaves and K+, Ca2+, Mg2+, Zn2+content in roots significantly decreased, too. However, Exogenous EBR increased the contents of the free Spm, conjugated Spd, Spm and bound Put, Spd, Spm. Meanwhile, EBR further increased the PAs content and (Spd+Spm)/Put ratio and reduced the Put/PAs ratio. Ca2+, Mg2+, Fe3+, Zn2+and Mn2+content in leaves and Ca2+, Mg2+, Fe3+, Mn2+content in roots significantly increased, too. Thus, EBR not only could regulate the formation and contents of polyamines but also promote the absorption of nutrients, make the leaf keep a higher photosynthetic performance, and alleviate the damage of hypoxic stress.